Human immunodeficiency virus antigens, vectors, compositions, and methods of use thereof

ABSTRACT

Synthetic HIV envelope proteins, vectors and compositions thereof, and methods for inducing protective immunity against human immunodeficiency virus (HIV) infection are described. Viral expression vectors encoding the synthetic HIV envelope proteins can be used in vaccines to provide improved protective immunity against HIV.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus (HIV) affects millions of people worldwide,and the prevention of HIV through an efficacious vaccine remains a veryhigh priority, even in an era of widespread antiretroviral treatment.HIV-1 is the most common and pathogenic strain of the virus, with morethan 90% of HIV/AIDS cases deriving from infection with HIV-1 group M.The M group is subdivided further into clades or subtypes. Anefficacious vaccine ideally would be capable of eliciting both potentcellular responses and broadly neutralizing antibodies capable ofneutralizing HIV-1 strains from different clades.

The high genetic variability of HIV-1 makes the development of a HIV-1vaccine an unprecedented challenge. In order to improve coverage ofpotential T-cell epitopes, and improve cellular responses, “mosaic”HIV-L Gag, Pol and Env antigens, derived from HIV Group Antigen (Gag),Polymerase (Pol), and Envelope (Env) proteins, were described by othersand developed in an attempt to provide maximal coverage of potentialT-cell epitopes (e.g., Barouch et al, Nat Med 2010, 16: 319-323). Themosaic antigens are similar in length and domain structure to wild-type,naturally occurring HIV-1 antigens.

For example, mosaic HIV antigens described and used in vaccines includethose described in Barouch et al, supra, and WO 2010/059732 such as:

-   -   (a) Gag mosaic antigens including:        -   (a)(i) a first mosaic Gag sequence (“mos1Gag”) having the            amino acid sequence as set forth herein in SEQ ID NO: 1, and    -   (a) (ii) a second mosaic Gag sequence (“mos2Gag”) having the        amino acid sequence as set forth herein in SEQ ID NO: 2;    -   (b) Pol mosaic antigens including:        -   (b)(i) a first mosaic Pol sequence (“moslPol”) having the            amino acid sequence as set forth herein in SEQ ID NO: 3, and        -   (b)(ii) a second mosaic Pol sequence (“mos2Pol”) having the            amino acid sequence as set forth herein in SEQ ID NO: 4; and    -   (c) Env mosaic antigens including:        -   (c)(i) a first mosaic Env sequence (“mos1Env”) having the            amino acid sequence as set forth herein in SEQ ID NO: 5, and        -   (c)(ii) a second mosaic Env sequence (“mos2Env”) having the            amino acid sequence as set forth herein in SEQ ID NO: 6.

Sequences encoding these antigens have been cloned in vectors, forexample, such as recombinant adenoviral vectors, e.g., recombinantadenovirus serotype 26 (rAd26), and these recombinant vectors werepreviously used as vaccines to generate immune responses to the antigens(see e.g. Barouch et al, supra; and WO 2010/059732). For example, themos1Gag and mos1Pol mosaic antigen sequences are typically combined intoa fusion protein of Gag and Pol (“mos1GagPol”), and the coding sequenceof which is cloned into a first Ad26 vector (“rAd26.mos1GagPol”); andthe mos2Gag and mos2Pol antigen sequences are combined into anotherfusion protein of Gag and Pol (“mos2GagPol”), and the coding sequence ofwhich is cloned into a second Ad26 vector (“rAd26.mos2GagPol”).Constructs encoding mos1Env and mos2Env are typically cloned intoseparate Ad26 vectors (“rAd26.mos1Env” and “rAd26.mos2Env”,respectively).

A set of such mosaic antigens as described above gives good globalcoverage of Group M HIV-1 isolates, where rAd26 vectors encoding mosaic1 antigen sequences (e.g., rAd26.mos1GagPol and rAd26.mos1Env) favorclade B and CRF01 HIV-1 subtypes, and rAd26 vectors encoding mosaic 2antigen sequences (e.g., rAd26.mos2GagPol and rAd26.mos2Env) favor cladeC strains. Mosaic HIV-1 Gag, Pol, and Env antigens expressed in rAd26vectors can be used to improve both the breadth and depth ofantigen-specific T-lymphocyte responses in rhesus monkeys, withoutcompromising the magnitude of both cellular and humoral responses whencompared with consensus or natural sequence HIV-1 antigens (Barouch etat, supra; and WO 2010/059732).

However, upon further development efforts on the vaccine componentsdescribed above, it was found that rAd26.mos2Env showed non-optimal cellsurface expression and immune response in non-human primates, butmoreover displayed a hitherto unreported, unexpected and unpredictablenon-optimal genetic stability during the manufacturing process ascompared to the other rAd26 vectors, such as rAd26.mos1Env. Thus,vaccines containing rAd26.mos2Env may result in non-optimal immuneresponses against Clade C HIV-1 subtypes, since the mos2Env mosaicantigen favors clade C HIV-1 strains. Accordingly, there is a need foran alternative to the mos2Env antigen in vaccines against HIV that canbe used to induce improved immune responses against HIV-1 clade C.

BRIEF SUMMARY OF THE INVENTION

The invention relates to novel synthetic human immunodeficiency virus(HIV) envelope proteins that have improved cell surface expression andgenetic stability as compared to the previously described mos2Envantigen. The invention also relates to compositions and methods of usingsuch novel synthetic HIV envelope proteins and/or coding sequencesthereof to induce increased immune responses against HIV-1, particularlyHIV-1 clade C, preferably when used in combination with other HIVantigens.

In one general aspect, the invention relates to a nucleic acid encodinga synthetic HIV envelope protein comprising the amino acid sequence ofSEQ ID NO: 8, or SEQ ID NO:8 having one or more mutations selected fromthe group consisting of (i) I529P (i.e., a substitution of lie to Pro atposition 529 of SEQ ID NO:8), (ii) K480E (i.e., a substitution of Lys toGlu at position 480 of SEQ ID NO:8), and (iii) a combination ofEK479-480RRRR (i.e. a replacement of Glu-Lys at positions 479-480 of SEQID NO:8 with four consecutive Arg residues), I529P, A471C (i.e., asubstitution of Ala to Cys at position 471 of SEQ ID NO:8) and T575C(i.e., a substitution of Thr to Cys at position 575 of SEQ ID NO:8). Inone embodiment, the synthetic HIV envelope protein further comprises asignal sequence, for instance a signal sequence having the amino acidsequence selected from the group consisting of SEQ ID NOs: 9-12. In oneembodiment, the signal sequence has the amino acid sequence of SEQ IDNO: 9.

In certain embodiments, the synthetic HIV envelope protein furthercomprises a transmembrane domain, preferably a transmembrane domainhaving the amino acid sequence of SEQ ID NO: 13.

In certain embodiments, the synthetic HIV envelope protein furthercomprises a fragment of a cytoplasmic domain, preferably a fragment of acytoplasmic domain comprising the amino acid sequence of SEQ ID NO:14,or amino acids 1-4 thereof (i.e., NRVR). In embodiments wherein thesynthetic HIV envelope protein further comprises a transmembrane domainand a fragment of a cytoplasmic domain, it is preferred that the proteinalso comprises the amino acid sequence of SEQ ID NO: 37, which is fusedto the carboxyl-terminus (C-terminus) of SEQ ID NO:8 and theamino-terminus (N-terminus) of the transmembrane region.

In another embodiment, the synthetic HIV envelope protein comprises atrimerization domain, for instance, a trimerization domain comprisingthe amino acid sequence of SEQ ID NO: 15 (GCN4) or SEQ ID NO:16 (foldondomain). In one preferred embodiment, the trimerization domain comprisesthe amino acid sequence of SEQ ID NO: 15. Such embodiments withtrimerization domains are useful for soluble (i.e. non membrane-bound)synthetic HIV envelope proteins based on the ectodomain sequencesprovided herein, such as that comprising the amino acid sequence of SEQID NO: 8, wherein the trimerization domain is located at the C-terminusof the synthetic HIV envelope protein.

In yet other embodiments, the synthetic HIV envelope protein comprisesSEQ ID NO: 8 with the following mutations: EK479-480RRRR, I529P, A471Cand T575C. The introduction of 6 consecutive arginine residues(positions 478 and 481 in the native sequence of SEQ ID NO: 8 alreadyare Arg residues) results in a further optimized furin cleavage site, sothat an improved processed (i.e. cleaved) ectodomain is obtained. Thethree mutations of I529P, A471C and T575C are known as SOSIP mutations,wherefrom the last two mutations result in introduction of a possibledisulfide bridge between the newly created cysteine residues. Overall,these mutations result in a soluble, trimerized, synthetic HIV envelopeprotein, without necessity for a trimerization domain.

In a preferred embodiment, the invention relates to a nucleic acidencoding a synthetic HIV envelope protein comprising the amino acidsequence of SEQ TD NO: 17, SEQ ID NO: 18, or as 1-686 of SEQ ID NO: 19.Most preferably the synthetic HIV envelope protein encoded by thenucleic acid comprises or consists of the amino acid sequence of SEQ IDNO: 18.

In another general aspect, the invention relates to a vector comprisinga nucleic acid encoding a synthetic HIV envelope protein according to anembodiment of the invention. In one embodiment, the vector is a viralvector. In a preferred embodiment, the viral vector is an adenoviralvector. In one preferred embodiment, the adenoviral vector is anadenovirus 26 vector.

Another general aspect of the invention relates to a composition,preferably a vaccine composition, comprising an immunogenicallyeffective amount of a vector according to an embodiment of theinvention, and a carrier, wherein the nucleic acid encoding thesynthetic HIV envelope protein is operably linked to a promotersequence. In one embodiment, the composition comprises an adenovirusvector, preferably an adenovirus 26 vector, encoding a synthetic HIVenvelope protein comprising the amino acid sequence of SEQ ID NO: 18.

In another general aspect, the invention relates to a vaccinecombination for inducing an immune response against a humanimmunodeficiency virus (HIV) in a subject in need thereof. The vaccinecombination comprises a first composition comprising an immunogenicallyeffective amount of a vector, preferably an adenovirus vector, morepreferably an adenovirus 26 vector, encoding a synthetic HIV envelopeprotein having the amino acid sequence of SEQ ID NO: 18, a secondcomposition comprising an immunogenically effective amount of a secondvector, preferably a second adenovirus vector, more preferably a secondadenovirus 26 vector, encoding an HIV antigenic polypeptide comprisingthe amino acid sequence of SEQ ID NO: 5, and optionally at least oneadditional composition comprising an immunogenically effective amount ofat least one selected from the group consisting of a vector encoding anantigenic polypeptide having the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-4, 28 and 29, and a polypeptidecomprising an immunogenically effective amount of an isolated HIVantigenic polypeptide, including but not limited to, a polypeptidehaving residues 30-708 of the amino acid sequence of SEQ ID NO: 7, or apolypeptide having residues 30-724 of SEQ ID NO:36, wherein the firstcomposition, second composition and optional additional composition arepresent in the same composition or in one or more differentcompositions.

Yet another general aspect of the invention relates to methods ofinducing an immune response against a human immunodeficiency virus (HIV)in a subject in need thereof, comprising administering to the subject acomposition or vaccine combination according to an embodiment of theinvention. The invention also relates to methods of inducing an immuneresponse against an HIV comprising priming and boosting the immuneresponse using a composition or a vaccine combination according to anembodiment of the invention.

Yet a further aspect of the invention relates to a synthetic HIVenvelope protein comprising the amino acid sequence of SEQ ID NO: 8, orSEQ ID NO: 8 having one or more mutations selected from the groupconsisting of (i) I529P, (ii) K480E, (iii) a combination ofEK479-480RRRR, I529P, A471C and T575C. In one embodiment, the syntheticHIV envelope protein comprises SEQ ID NO:8 with the mutations ofEK479-480RRRR, I529P, A471C and T575C. In another embodiment, thesynthetic HIV envelope protein comprises residues 30-704 or 30-711 ofthe amino acid sequence of SEQ ID NO: 18. In yet another embodiment thesynthetic HIV envelope protein comprises residues 30-686 of the aminoacid sequence of SEQ ID NO:19.

Another aspect of the invention relates to a cell, preferably anisolated cell, comprising a vector according to an embodiment of theinvention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood that the invention is notlimited to the precise embodiments shown in the drawings.

In the drawings:

FIGS. 1A-IC are schematic representations of the structure of HIVenvelope proteins; FIG. 1A shows a full length HIV envelope protein;FIG. 1B shows the structure of a soluble single chain HIV envelopeprotein according to an embodiment of the invention in which thetransmembrane domain (TM) is replaced with a GCN4 trimerization domain,and the furin cleavage site is mutated (sC4); FIG. 1C shows thestructure of a membrane bound HIV envelope protein according to anembodiment of the invention comprising a transmembrane domain and afragment of a cytoplasmic domain (C4D7);

FIG. 2 shows expression levels of the soluble sC1 envelope protein,which is based on the mos2Env mosaic antigen sequence with an additionalC-terminal trimerization domain, and a soluble synthetic HIV envelopeprotein (sC4) according to an embodiment of the invention; expressionwas measured by quantitative Western blot using a polyclonal antibodyagainst gp120; plasmids encoding sC1 or sC4 were transiently expressedtwice, and each transfection was quantified twice by densitometry; thesC1 protein showed very low expression levels compared to the sC4synthetic HIV envelope protein, which showed relatively high expressionlevels;

FIGS. 3A and 3B show the binding of synthetic HIV envelope proteins withmonoclonal antibody 17b (mAb17b) in the presence (light gray) andabsence (dark gray) of soluble CD4 as determined by ELISA assay; FIG. 3Ashows binding of sC1; FIG. 3B shows binding of sC4;

FIG. 4 is an image of a Western blot from a native polyacrylamide gelelectrophoresis of the sC1 protein, and the sC4 synthetic HIV envelopeprotein;

FIG. 5 shows the relative cell surface expression levels of themembrane-bound C1, C1D7, C4 and C4D7 synthetic HIV envelope proteins byFACS analysis of cells expressing these proteins using an anti-gp120polyclonal antibody (GP120), and by binding to broadly neutralizingantibodies PG9 (PG9) and PG16 (PG16) that are quaternary-structuredependent and preferentially bind to correctly folded Env trimer;

FIG. 6 is a graphical representation of the stability of adenovirusvectors containing sequences encoding synthetic HIV envelope proteins ofthe invention including full-length C4 (FLC4), C4D7, and sC4 aftermultiple viral passages; recombinant adenovirus 26 vectors weregenerated in PER.C6 cells; after the initial 3 passages for transfectionand plaque purification, 5 plaques were selected and upscaled for 10passages in T25 format, resulting in a total viral passage number (vpn)of 13; the stability after vpn 3, 5, 10, and 13 as determined by E1transgene cassette polymerase chain reaction (PCR) is shown; forexample, 315 means 3 plaques were stable out of 5 plaques tested, and515 means 5 plaques were stable out of 5 plaques tested; and

FIGS. 7A and 7B show virus neutralization titers against HIV-1 envelopepseudotyped virus particles (EVPs) in a TZM-b1 cell-based neutralizationassay in rabbits; log 10-transformed IC₅₀ values of the high-adenoviralvector dosed groups were measured against EVPs VSV-G (negative control)and MW965.26 (Tier 1A clade C) at weeks 1, 8, 14, and 20; each dotrepresents the log 10-transformed IC₅₀ value of an individual rabbit,with the group mean indicated by a horizontal line; HD: Highest Dilutiontested (upper solid line); LD: Lowest Dilution tested (lower solidline); LOB: limit of background, 95 percentile value of compilednegative samples (dotted line); Log 10 IC₅₀ values exceeding the LD orHD threshold were set at the corresponding line; a one-waynon-parametric comparison with control using the Dunn method for jointranking was done for each time point; statistically significantdifferences are indicated in the graphs: *=P<0.05, **=P<0.01, and***=P<0.001; FIG. 7A shows the results with VSV-G (negative control);and FIG. 7B shows the results with MW965.26 (Tier 1A clade C).

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification. All patents,published patent applications and publications cited herein areincorporated by reference as if set forth fully herein. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

As used herein, “subject” means any animal, preferably a mammal, mostpreferably a human, to whom will be or has been administered a vector,composition or combination vaccine according to embodiments of theinvention. The term “mammal” as used herein, encompasses any mammal.Examples of mammals include, but are not limited to, cows, horses,sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys,humans, etc., more preferably a human.

The invention generally relates to synthetic HIV envelope proteins,nucleic acid and vectors encoding the synthetic HIV envelope proteins,and methods of inducing an immune response against HIV with vectorsencoding the synthetic HIV envelope proteins or the synthetic HIVenvelope proteins, alone or in combination with one or more additionalvectors encoding one or more additional HIV antigenic polypeptidesand/or in combination with one or more additional isolated HIV antigenicpolypeptides.

Human immunodeficiency virus (HIV) is a member of the genusLentivirinae, which is part of the family of Retroviridae. Two speciesof HIV infect humans: HIV-1 and HIV-2. HIV-1 is the most common strainof HIV virus, and is known to be more pathogenic than HIV-2. As usedherein, the terms “human immunodeficiency virus” and “HIV” refer, butare not limited to, HIV-1 and HIV-2.

HIV is categorized into multiple clades with a high degree of geneticdivergence. As used herein, the term “HIV clade” or “HIV subtype” refersto related human immunodeficiency viruses classified according to theirdegree of genetic similarity. There are currently three groups of HIV-1isolates: M, N and O. Group M (major strains) consists of at least tenclades, A through J. Group O (outer strains) can consist of a similarnumber of clades. Group N is a new HIV-isolate that has not beencategorized in either group M or O.

As used herein, the terms “HIV antigenic polypeptide,” “HIV antigenicprotein,” and “HIV immunogen” refer to a polypeptide capable of inducingan immune response, e.g., a humoral and/or cellular mediated response,against HIV in a subject. The antigenic polypeptide can be a protein ofthe HIV, a fragment or epitope thereof, or a combination of multiple HIVproteins or portions thereof, that can induce an immune response orproduce an immunity, e.g., protective immunity, against the HIV in asubject.

Preferably, an antigenic polypeptide is capable of raising in a host aprotective immune response, e.g., inducing an immune response against aviral disease or infection, and/or producing an immunity in (i.e.,vaccinates) a subject against a viral disease or infection, thatprotects the subject against the viral disease or infection. Forexample, the antigenic polypeptide can comprise a protein or fragmentsthereof from Simian Immunodeficiency Virus (SIV) or an HIV, such as theHIV or SIV envelope gp160 protein, the HIV or SIV matrix/capsidproteins, and the HIV or SIV gag, pol and env gene products.

An HIV antigenic polypeptide can be any HIV-1 or HIV-2 antigen orfragment thereof. Examples of HIV antigens include, but are not limitedto gag, pol, and env gene products, which encode structural proteins andessential enzymes. Gag, pol, and env gene products are synthesized aspolyproteins, which are further processed into multiple other proteinproducts. The primary protein product of the gag gene is the viralstructural protein gag polyprotein, which is further processed into MA,CA, SP1, NC, SP2, and P6 protein products. The pol gene encodes viralenzymes (Pol, polymerase), and the primary protein product is furtherprocessed into RT, RNase H, 1N, and PR protein products. The env geneencodes structural proteins, specifically glycoproteins of the virionenvelope. The primary protein product of the env gene is gp160, which isfurther processed into gp120 and gp41. Other examples of HIV antigensinclude gene regulatory proteins Tat and Rev; accessory proteins Nef,Vpr, Vif and Vpu; capsid proteins, nucleocapsid proteins, and p24 viralprotein.

In certain embodiments, the HIV antigenic polypeptide comprises an HIVGag, Env, or Pol antigen, or any antigenic portion or epitope orcombination thereof, preferably an HIV-1 Gag, Env, or Pol antigen or anyantigenic portion or epitope or combination thereof.

HIV antigenic polypeptides can also be mosaic HIV antigens. As usedherein, “mosaic antigen” refers to a recombinant protein assembled fromfragments of natural sequences. Mosaic antigens resemble naturalantigens, but are optimized to maximize the coverage of potential T-cellepitopes found in the natural sequences, which improves the breadth andcoverage of the immune response. Mosaic HIV antigens for use with theinvention are preferably mosaic Gag, Pol, and/or Env antigens, and morepreferably a mosaic HIV-1 Gag, Pol, and/or Env antigens. As used herein,“a mosaic HIV Gag, Pol, and/or Env antigen” specifically refers to amosaic antigen comprising multiple epitopes derived from one or more ofthe Gag, Pol and/or Env polyprotein sequences of HIV.

In one embodiment, a mosaic HIV antigen for use with the invention is amosaic HIV Gag antigen with epitopes derived from the sequences of gaggene products (examples are provided in SEQ ID NOs: 1, 2); a mosaic HIVPol antigen with epitopes derived from the sequences of pol geneproducts (examples are provided in SEQ ID NOs: 3, 4); or a mosaic HIVEnv antigen with epitopes derived from the sequences of env geneproducts (examples are provided in SEQ ID NOs: 5, 6; also the novelantigens of the invention, e.g. in SEQ ID NOs: 8, 17, 18, 19, can beconsidered mosaic HIV Env antigens). In certain embodiments, a mosaicHIV antigen for use with the invention may comprise a combination ofepitopes derived from sequences of gag, pol, and/or env gene products.Illustrative and non-limiting examples include mosaic Env-Pol antigenswith epitopes derived from the sequences of env and pol gene products;mosaic Gag-Pol antigens with epitopes derived from the sequences of gagand poi gene products; and mosaic Gag-Env antigens with epitopes derivedfrom the sequences of gag and env gene products. The sequences of gag,pol, and env gene products can be derived from one or more clades.

Examples of mosaic HIV Gag, Pol and/or Env antigens that can be used inthe invention include those described in, e.g., US20120076812; Barouchet al., Nat Med 2010, 16:319-323; and Barouch et al., Cell 155:1-9,2013, all of which are incorporated herein by reference in theirentirety. Preferably, mosaic HIV Gag, Pol, and/or Env antigens for usewith the present invention include, but are not limited to, mos1Gag (SEQID NO: 1), mos2Gag (SEQ ID NO: 2), moslPol (SEQ ID NO: 3), mos2Pol (SEQID NO: 4), mos1Env (SEQ ID NO: 5), mos2Env (SEQ ID NO: 6), mos1GagPol(SEQ ID NO: 28), mos2GagPol (SEQ ID NO: 29), and combinations thereof.

As used herein, each of the terms “HIV envelope protein,” “env protein,”and “Env” refers to a protein that is expressed on the envelope of anHIV virion and enables an HIV to target and attach to the plasmamembrane of HIV infected cells, or a fragment or derivative thereof thatcan induce an immune response or produce an immunity against the HIV ina subject in need thereof. The HIV env gene encodes the precursorprotein gp160, which is proteolytically cleaved into the two matureenvelope glycoproteins, gp120 and gp41. The cleavage reaction ismediated by a host cell protease, furin, at a sequence highly conservedin retroviral envelope glycoprotein precursors. More specifically, gp160trimerizes to (gp160), and then undergoes cleavage into the twononcovalently associated gp120 and gp41. Viral entry is subsequentlymediated by a trimer of gp120/gp41 heterodimers. Gp120 is the receptorbinding fragment, and binds to the CD4 receptor on a target cell thathas such a receptor, such as, e.g., a T-helper cell. Gp41, which isnoncovalently bound to gp120, is the fusion fragment and provides thesecond step by which HIV enters the cell. Gp41 is originally buriedwithin the viral envelope, but when gp120 binds to a CD4 receptor, gp120changes its conformation causing gp41 to become exposed, where it canassist in fusion with the host cell. Gp140 is the uncleaved ectodomainof trimeric gp160, i.e., (gp160)₃, that has been used as a surrogate forthe native state of the cleaved, viral spike.

According to embodiments of the invention, an “HIV envelope protein” canbe a gp160, gp140, gp120, gp41 protein, combinations, fusions,truncations or derivatives thereof. For example, an “HIV envelopeprotein” can include a gp120 protein noncovalently associated with agp41 protein. It can also include a stabilized trimeric gp140 proteinthat can have or can be modified to include a trimerization domain thatstabilizes trimers of gp140. Examples of trimerization domains include,but are not limited to, the T4-fibritin “foldon” trimerization domain;the coiled-coil trimerization domain derived from GCN4; and thecatalytic subunit of E. coli aspartate transcarbamoylase as a trimertag. An “HIV envelope protein” can also be a truncated HIV envelopeprotein including, but not limited to, envelope proteins comprising aC-terminal truncation in the ectodomain (i.e. the domain that extendsinto the extracellular space), a truncation in the gp41, such as atruncation in the transmembrane domain of gp41, or a truncation in thecytoplasmic domain of gp41. An “HIV envelope protein” can further be aderivative of a naturally occurring HIV envelope protein having sequencemutations, e.g., in the furin cleavage sites, and/or so-called SOSIPmutations.

Preferably, an “HIV envelope protein” is a “synthetic HIV envelopeprotein.” As used herein, the term “synthetic HIV envelope protein”refers to a non-naturally occurring HIV envelope protein that isoptimized to induce an immune response or produce an immunity againstone or more naturally occurring HIV strains in a subject in needthereof. Mosaic HIV Env proteins are examples of synthetic HIV Envproteins, and the invention provides novel synthetic HIV Env antigens,e.g. the ones comprising SEQ ID NOs: 8, 17, 18, or 19.

Synthetic HIV Envelope Proteins and Coding Sequences Thereof

Embodiments of the invention relate to novel synthetic HIV envelopeproteins and nucleic acid molecules encoding these.

In one embodiment, the invention relates to a synthetic HIV envelopeprotein comprising the amino acid sequence of SEQ ID NO: 8, or SEQ IDNO:8 having one or more mutations selected from the group consisting of(i) I529P, (ii) K480E, and (iii) a combination of EK479-480RRRR, I529P,A471C and T575C. SEQ ID NO:8 comprises a synthetic mature gp120 and asynthetic truncated gp4 without the transmembrane region, nor thecytoplasmic domain. SEQ ID NO:8 is a non-naturally occurring sequencecomprised of a chimera of sequences from the mos2Env mosaic antigen (SEQID NO: 6), and other HIV envelope protein sequences. The sequence of thenovel synthetic Env antigen comprising SEQ ID NO:8 is optimized toprovide broad coverage and an enhanced T-cell response against HIV cladeC (as compared to the mos2Env antigen (SEQ ID NO: 6)). In certainembodiments, further amino acids can be added to SEQ ID NO: 8 or one ofits variants defined herein.

In certain embodiments, the synthetic HIV envelope protein furthercomprises a signal sequence. The synthetic HIV envelope protein issynthesized with a signal sequence that is cleaved from the nascentpolypeptide chain during its transport into the lumen of the endoplasmicreticulum (ER). In principle, any known signal sequence could be used.Preferably an HIV Env signal sequence or a variant thereof is used.Different signal sequences have been used in the art for HIV Envproteins (see e.g. WO 2014/107744). In certain embodiments, the signalsequence comprises SEQ ID NO:9, SEQ ID NO: 10. SEQ ID NO:11 or SEQ IDNO:12. In one preferred embodiment, the signal sequence comprises SEQ IDNO: 9.

In certain embodiments, the synthetic HIV envelope protein furthercomprises a transmembrane domain. The transmembrane domain anchors thesynthetic HIV envelope protein to the ER membrane, and contributes tomembrane assembly and function of the HIV envelope. Preferably, thetransmembrane domain comprises SEQ ID NO:13.

In another embodiment, the synthetic HIV envelope protein comprises agp41 having a truncated cytoplasmic domain. The gp41 has an unusuallylong cytoplasmic domain at its carboxyl end, typically about 150 aminoacids (Edwards et al., J. Virology, 2002, 76:2683-2691). Truncation ofthe cytoplasmic domain was reported to induce exposure of conservedregions in the ectodomain of HIV-1 Env protein (Id.). The truncatedcytoplasmic domain in a synthetic HIV envelope of the invention canrange from one to about 140 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140amino acids of a full-length cytoplasmic domain. In certain embodimentsthe truncated cytoplasmic domain is derived from amino acids 704-862 ofSEQ ID NO: 17 (i.e. from the cytoplasmic domain of the C4 molecule ofthe invention), by truncation after a given amino acid up to theC-terminus. In a preferred embodiment, the synthetic HIV envelopeprotein comprises a truncated cytoplasmic domain having 1 to 10 aminoacids residues, more preferably 4 to 8 amino acid residues, and mostpreferably 7 amino acid residues of an HIV gp41 cytoplasmic domain. Thecytoplasmic domain or fragment thereof of a synthetic HIV envelopeprotein is located C-terminal to the extracellular domain (ectodomain),and when the synthetic HIV envelope protein also comprises atransmembrane domain, the cytoplasmic domain or fragment thereof islocated C-terminal to the transmembrane domain. See, e.g., FIGS. 1A and1C. In a particular embodiment, the synthetic HIV envelope proteincomprises a gp41 with a truncated cytoplasmic domain having the aminoacid sequence of SEQ ID NO:14 or a fragment thereof, such as residues1-4 thereof (i.e. NRVR). Other truncated cytoplasmic domains have beendescribed and could be used (e.g. Schiernle et al., PNAS 1997;Abrahamyan et al., J Virol 2005).

In embodiments wherein the synthetic HIV envelope protein furthercomprises a transmembrane domain and a fragment of a cytoplasmic domain,it is preferred that the protein also comprises the amino acid sequenceof SEQ ID NO: 37, which contains residues 655-682 of SEQ ID NO: 18,wherein the amino acid sequence of SEQ ID NO: 37 is fused to theC-terminus of SEQ ID NO: 8 and the N-terminus of the transmembranedomain.

In a particularly preferred embodiment of the invention, the syntheticHIV envelope protein further comprises a transmembrane domain, such asthat having the amino acid sequence of SEQ ID NO:13, and a truncatedcytoplasmic domain or a fragment of cytoplasmic domain, such as thathaving the amino acid sequence of SEQ ID NO: 14 or residues 1-4 of SEQID NO:14 (i.e., NRVR). Most preferably, the synthetic HIV envelopeprotein comprises or consists of the amino acid sequence of SEQ ID NO:18, with or without the signal sequence (i.e., amino acid resides 1-29of SEQ ID NO: 18).

In another embodiment, the synthetic HIV envelope protein comprises atrimerization domain that replaces an Env transmembrane region. Thetrimerization domain increases the stability of an Env trimericstructure. Preferably, the synthetic HIV envelope protein comprises agp140 polypeptide that is modified to include a trimerization domainthat stabilizes trimers of gp140. Examples of trimerization domainsinclude, but are not limited to, the T4-fibritin “foldon” trimerizationdomain, such as that comprising the amino acid sequence of SEQ ID: 16;the coiled-coil trimerization domain derived from GCN4, such as thatcomprising the amino acid sequence of SEQ ID: 15; the catalytic subunitof E. coli aspartate transcarbamoylase as a trimer tag; ormatrillin-based trimerization motifs. If present, the trimerizationdomain typically is located C-terminal to the extracellular domain (seeFIG. 1B). In certain preferred embodiments where the synthetic HIVenvelope protein comprises a trimerization domain, the synthetic HIVenvelope protein comprises the amino acid sequence of SEQ ID NO: 19,with or without the signal sequence (i.e., amino acid residues 1-29 ofSEQ ID NO: 19). These embodiments with trimerization domains are mainlyuseful for soluble ectodomain variants of the synthetic HIV envelopeprotein. In certain embodiments of such soluble variants of theinvention, it is possible to mutate the furin cleavage site (e.g.mutation of Lys to Glu at position 480 in SEQ ID NO: 8) to inactivatethis cleavage site, so that the protein will be a single chain, thiscombines well with a trimerization domain, especially with the GCN4trimerization domain of SEQ ID NO: 19.

Alternative versions of such soluble ectodomain variants of thesynthetic HIV envelope protein without use of trimerization domains arealso embodiments of the invention, and can be prepared from SEQ ID NO: 8by combining mutations that optimize the furin cleavage site (replacingthe Gly-Lys dipeptide at positions 479-480 by four Arg residues) as wellas so-called SOSIP mutations (I>P mutation at position 529, andintroduction of a disulfide bridge between positions 471 and 575 byreplacement of the respective Ala and Thr on those positions in SEQ IDNO: 8 each with a Cys residue). This yields a protein having the aminoacid sequence of SEQ ID NO: 8 with the following combination ofmutations: EK479-480RRRR, I529P, A471C and T575C.

One possible modification to further increase the trimer content of asynthetic HIV envelope protein of the invention (comprising SEQ ID NO:8), is modification of lie to Pro at position 529. This can be effectivefor both soluble and membrane-bound variants.

Vectors

Another general aspect of the invention relates to vectors comprisingnucleic acid encoding a synthetic HIV envelope protein. According toembodiments of the invention, the vectors can comprise any of thesynthetic HIV envelope proteins described herein. In a preferredembodiment of the invention, the vector comprises nucleic acid encodinga synthetic HIV envelope protein comprising the amino acid sequence ofSEQ ID NO: 8, SEQ ID NO:17, SEQ ID NO: 18, or SEQ ID NO: 19, and morepreferably SEQ ID NO: 18.

According to embodiments of the invention, the nucleic acid encoding thesynthetic HIV envelope protein is operably linked to a promoter, meaningthat the nucleic acid is under the control of a promoter. The promotercan be a homologous promoter (i.e., derived from the same genetic sourceas the vector) or a heterologous promoter (i.e., derived from adifferent vector or genetic source). Examples of suitable promotersinclude the cytomegalovirus (CMV) promoter and the Rous Sarcoma virus(RSV) promoter. Preferably, the promoter is located upstream of thenucleic acid within an expression cassette. An exemplary CMV promotersequence that can be operably linked to nucleic acid encoding thesynthetic HIV envelope protein is shown in SEQ ID NO: 24.

According to embodiments of the invention, a vector can be an expressionvector. Expression vectors include, but are not limited to, vectors forrecombinant protein expression and vector for delivery of nucleic acidinto a subject for expression in a tissue of the subject, such as aviral vector. Examples of viral vectors suitable for use with theinvention include, but are not limited to adenoviral vectors,adeno-associated virus vectors, pox virus vectors, MVA vectors, entericvirus vectors, Venezuelan Equine Encephalitis virus vectors, SemlikiForest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors,etc. The vector can also be a non-viral vector. Examples of non-viralvectors include, but are not limited to plasmids, bacterial artificialchromosomes, yeast artificial chromosomes, bacteriophages, etc.

In certain embodiments of the invention, the vector is an adenovirusvector. An adenovirus according to the invention belongs to the familyof the Adenoviridae, and preferably is one that belongs to the genusMastadenovirus. It can be a human adenovirus, but also an adenovirusthat infects other species, including but not limited to a bovineadenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g.CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or a simian adenovirus(which includes a monkey adenovirus and an ape adenovirus, such as achimpanzee adenovirus or a gorilla adenovirus). Preferably, theadenovirus is a human adenovirus (HAdV, or AdHu), or a simian adenovirussuch as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV). In theinvention, a human adenovirus is meant if referred to as Ad withoutindication of species, e.g. the brief notation “Ad26” means the same asHadV26, which is human adenovirus serotype 26. Also as used herein, thenotation “rAd” means recombinant adenovirus, e.g., “rAd26” refers torecombinant human adenovirus 26.

Most advanced studies have been performed using human adenoviruses, andhuman adenoviruses are preferred according to certain aspects of theinvention. In certain preferred embodiments, a recombinant adenovirusaccording to the invention is based upon a human adenovirus. Inpreferred embodiments, the recombinant adenovirus is based upon a humanadenovirus serotype 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According toa particularly preferred embodiment of the invention, an adenovirus is ahuman adenovirus of serotype 26. An advantage of this serotypes is a lowseroprevalence and/or low pre-existing neutralizing antibody titers inthe human population, and experience with use in human subjects inclinical trials.

Simian adenoviruses generally also have a low seroprevalence and/or lowpre-existing neutralizing antibody titers in the human population, and asignificant amount of work has been reported using chimpanzee adenovirusvectors (e.g. U.S. Pat. No. 6,083,716; WO 2005/071093 WO 2010/086189; WO2010085984; Farina et al, 2001, J Virol 75; 11603-13 [13]; Cohen et al,2002, J Gen Virol 83: 151-55 [69]; Kobinger et al, 2006, Virology 346:394-401 [70]; Tatsis et al., 2007, Molecular Therapy 15: 608-17 [71];see also review by Bangari and Mittal, 2006, Vaccine 24: 849-62 [72];and review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39 [73]). Hence,in other embodiments, the recombinant adenovirus according to theinvention is based upon a simian adenovirus, e.g. a chimpanzeeadenovirus. In certain embodiments, the recombinant adenovirus is basedupon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1,29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44,45, 46, 48, 49, 50 or SA7P.

Preferably, the adenovirus vector is a replication deficient recombinantviral vector, such as rAd26, rAd35, rAd48, rAd5HVR48, etc.

In a preferred embodiment of the invention, the adenoviral vectorscomprise capsid proteins from rare serotypes including Ad26. In thetypical embodiment, the vector is an rAd26 virus. An “adenovirus capsidprotein” refers to a protein on the capsid of an adenovirus (e.g., Ad26,Ad35, rAd48, rAd5HVR48 vectors) that is involved in determining theserotype and/or tropism of a particular adenovirus. Adenoviral capsidproteins typically include the fiber, penton and/or hexon proteins. Asused herein a “capsid protein” for a particular adenovirus, such as an“Ad26 capsid protein” can be, for example, a chimeric capsid proteinthat includes at least a part of an Ad26 capsid protein. In certainembodiments, the capsid protein is an entire capsid protein of Ad26. Incertain embodiments, the hexon, penton and fiber are of Ad26.

One of ordinary skill in the art will recognize that elements derivedfrom multiple serotypes can be combined in a single recombinantadenovirus vector. Thus, a chimeric adenovirus that combines desirableproperties from different serotypes can be produced. Thus, in someembodiments, a chimeric adenovirus of the invention could combine theabsence of pre-existing immunity of a first serotype withcharacteristics such as temperature stability, assembly, anchoring,production yield, redirected or improved infection, stability of the DNAin the target cell, and the like.

In certain embodiments the recombinant adenovirus vector useful in theinvention is derived mainly or entirely from Ad26 (i.e., the vector isrAd26). In some embodiments, the adenovirus is replication deficient,e.g., because it contains a deletion in the E1 region of the genome. Foradenoviruses being derived from non-group C adenovirus, such as Ad26 orAd35, it is typical to exchange the E4-orf6 coding sequence of theadenovirus with the E4-orf6 of an adenovirus of human subgroup C such asAd5. This allows propagation of such adenoviruses in well-knowncomplementing cell lines that express the E1 genes of Ad5, such as forexample 293 cells, PER.C6 cells, and the like (see, e.g. Havenga, etal., 2006, J Gen Virol 87: 2135-43; WO 03/104467). However, suchadenoviruses will not be capable of replicating in non-complementingcells that do not express the E1 genes of Ad5.

The preparation of recombinant adenoviral vectors is well known in theart. Preparation of rAd26 vectors is described, for example, in WO2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplarygenome sequences of Ad26 are found in GenBank Accession EF 153474 and inSEQ ID NO:1 of WO 2007/104792. Examples of vectors useful for theinvention for instance include those described in WO2012/082918, thedisclosure of which is incorporated herein by reference in its entirety.

Typically, a vector useful in the invention is produced using a nucleicacid comprising the entire recombinant adenoviral genome (e.g., aplasmid, cosmid, or baculovirus vector). Thus, the invention alsoprovides isolated nucleic acid molecules that encode the adenoviralvectors of the invention. The nucleic acid molecules of the inventioncan be in the form of RNA or in the form of DNA obtained by cloning orproduced synthetically. The DNA can be double-stranded orsingle-stranded.

The adenovirus vectors useful in the invention are typically replicationdeficient. In these embodiments, the virus is rendered replicationdeficient by deletion or inactivation of regions critical to replicationof the virus, such as the E1 region. The regions can be substantiallydeleted or inactivated by, for example, inserting a gene of interest,such as a gene encoding a synthetic HIV envelope protein (usually linkedto a promoter), or a gene encoding an HIV antigenic polypeptide (usuallylinked to a promoter) within the region. In some embodiments, thevectors of the invention can contain deletions in other regions, such asthe E2, E3 or E4 regions, or insertions of heterologous genes linked toa promoter within one or more of these regions. For E2- and/orE4-mutated adenoviruses, generally E2- and/or E4-complementing celllines are used to generate recombinant adenoviruses. Mutations in the E3region of the adenovirus need not be complemented by the cell line,since E3 is not required for replication.

A packaging cell line is typically used to produce sufficient amounts ofadenovirus vectors for use in the invention. A packaging cell is a cellthat comprises those genes that have been deleted or inactivated in areplication deficient vector, thus allowing the virus to replicate inthe cell. Suitable packaging cell lines for adenoviruses with a deletionin the E1 region include, for example, PER.C6, 911, 293, and E1 A549.

According to embodiments of the invention, and as noted above, any ofthe synthetic HIV envelope proteins described herein can be expressed inthe vectors of the invention. In view of the degeneracy of the geneticcode, the skilled person is well aware that several nucleic acidsequences can be designed that encode the same protein, according tomethods entirely routine in the art. The nucleic acid encoding thesynthetic HIV envelope protein can optionally be codon-optimized toensure proper expression in the treated host (e.g., human).Codon-optimization is a technology widely applied in the art. Somenon-limiting examples of sequences encoding a synthetic HIV envelopeprotein of the invention are provided in SEQ ID NOs: 25, 26 and 27.Typically, the nucleic acid encoding the synthetic HIV envelope proteinis cloned into the E1 and/or the E3 region of the adenoviral genome.

In a preferred embodiment of the invention, the vector is an adenovirusvector, and more preferably a rAd26 vector, most preferably a rAd26vector with at least a deletion in the E1 region of the adenoviralgenome, e.g. such as that described in Abbink, J Viol, 2007. 81(9): p.4654-63, which is incorporated herein by reference.

The invention also provides cells, preferably isolated cells, comprisingany of the vectors described herein. The cells can be used forrecombinant protein production, or for the production of viralparticles.

Embodiments of the invention thus also relate to a method of a making asynthetic HIV antigenic polypeptide. The method comprises transfecting ahost cell with an expression vector comprising nucleic acid encoding thesynthetic HIV antigenic polypeptide operably linked to a promoter,growing the transfected cell under conditions suitable for expression ofthe synthetic HIV antigenic polypeptide, and isolating the synthetic HIVantigentic polypeptide from the cell. The synthetic HIV antigenicpolypeptide can be isolated or collected from the cell by any methodknown in the art including affinity chromatography, etc. Techniques usedfor recombinant protein expression will be well known to one of ordinaryskill in the art in view of the present disclosure.

The invention also includes a method for manufacturing a vector encodinga synthetic HIV antigenic polypeptide of the invention, the methodcomprising culturing a cell that comprises the vector, to propagate andmultiply the vector during said culturing, and isolating the vector thatencodes the synthetic HIV antigenic polypeptide of the invention fromthe cell culture, e.g. from the cells, from the culture medium, or both.The vector may be further purified according to methods known in theart.

In certain embodiments, the invention provides a vector according to anembodiment of the invention comprising a nucleic acid encoding asynthetic HIV antigenic polypeptide, and in certain exemplaryembodiments the nucleic acid has a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 25, 26 and 27.

Compositions

In another general aspect, the invention relates to a compositioncomprising a vector comprising a nucleic acid encoding a synthetic HIVenvelope protein and a carrier. According to embodiments of theinvention, any of vectors described herein can be included in thecomposition. Preferably, the vector is a viral vector, more preferablyan adenovirus vector, and even more preferably an adenovirus 26 vector.In a preferred embodiment, a composition comprises an adenovirus vector,preferably an adenovirus 26 vector encoding a synthetic HIV envelopeprotein comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO:18, or SEQ ID NO: 19, and more preferably the amino acid sequence of SEQID NO: 18.

In one aspect, the invention provides a combination vaccine comprisingone or more vectors together comprising nucleic acid sequences encoding(i) a synthetic HIV envelope protein comprising the amino acid sequenceof SEQ ID NO: 8 (e.g. SEQ ID NO: 18 or 19) and (ii) a second HIVenvelope protein comprising the amino acid sequence of SEQ ID NO: 5. Thevectors may each be in a separate composition, or be combined in asingle composition. Both nucleic acids in the vector(s) are intended tobe administered to one subject, which will result in an immune responseto HIV that is broader than the immune response that would be obtainedupon administration of either vector alone. Both nucleic acid sequencescould also be present on one single vector.

According to embodiments of the invention, a composition comprises animmunogenically effective amount of a vector, such as a viral vector. Asused herein, “an immunogenically effective amount” or “immunologicallyeffective amount” means an amount of a composition sufficient to inducea desired immune effect or immune response in a subject in need thereof.In one embodiment, an immunogenically effective amount means an amountsufficient to induce an immune response in a subject in need thereof. Inanother embodiment, an immunogenically effective amount means an amountsufficient to produce immunity in a subject in need thereof, e.g.,provide a protective effect against a disease such as a viral infection.An immunogenically effective amount can vary depending upon a variety offactors, such as the physical condition of the subject, age, weight,health, etc.; the particular application, whether inducing immuneresponse or providing protective immunity; the specific recombinantvector administered; the immunogen or antigenic polypeptide encoded bythe recombinant vector administered; the specific antigenic polypeptideadministered; and the particular disease, e.g., viral infection, forwhich immunity is desired. An immunogenically effective amount canreadily be determined by one of ordinary skill in the an in view of thepresent disclosure.

As general guidance, an immunogenically effective amount when used withreference to a recombinant viral vector such as an adenviral vector canrange from about 10⁸ viral particles to about 10¹² viral particles, forexample 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² viral particles. Animmunogenically effective amount can be administered in a singlecomposition, or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 compositions (e.g., tablets, capsules or injectables),wherein the administration of the multiple capsules or injectionscollectively provides a subject with the immunogenically effectiveamount. In general, when used with reference to a polypeptide, such asan isolated antigenic polypeptide, an immunogenically effective amountcan range from, e.g. about 0.3 to about 3000 microgram (μg), e.g. 1-1000μg, e.g. 10-500 μg, e.g. about 10, 50, 100, 150, 200, 250, 300, 350,400, 450 or 500 μg. As a non-limiting example, it is possible to combineadministration of the vector encoding the synthetic HIV Env antigen ofthe invention (having SEQ ID NO: 8) with administration of an Envpolypeptide, e.g. 250 μg of HIV Clade C Env trimer protein having aminoacids 30-708 of SEQ ID NO: 7. It is also possible to administer animmunogenically effective amount to a subject, and subsequentlyadminister another dose of an immunogenically effective amount to thesame subject, in a so-called prime-boost regimen. This general conceptof a prime-boost regimen is well known to the skill person in thevaccine field. Further booster administrations can optionally be addedto the regimen, as needed.

Compositions of the invention further comprise a carrier. A carrier caninclude one or more pharmaceutically acceptable excipients such asbinders, disintegrants, swelling agents, suspending agents, emulsifyingagents, wetting agents, lubricants, flavorants, sweeteners,preservatives, dyes, solubilizers and coatings. The precise nature ofthe carrier or other material can depend on the route of administration,e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous,intramucosal (e.g., gut), intranasal or intraperitoneal routes. Forliquid injectable preparations, for example, suspensions and solutions,suitable carriers and additives include water, glycols, oils, alcohols,preservatives, coloring agents and the like. For solid oralpreparations, for example, powders, capsules, caplets, gelcaps andtablets, suitable carriers and additives include starches, sugars,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like. For nasal sprays/inhalant mixtures, the aqueoussolution/suspension can comprise water, glycols, oils, emollients,stabilizers, wetting agents, preservatives, aromatics, flavors, and thelike as suitable carriers and additives.

Compositions of the invention can be formulated in any matter suitablefor administration to a subject to facilitate administration and improveefficacy, including, but not limited to, oral (enteral) administrationand parenteral injections. The parenteral injections include intravenousinjection or infusion, intra-arterial injection, subcutaneous injection,intramuscular injection, and intra-articular injection. Compositions ofthe invention can also be formulated for other routes of administrationincluding transmucosal, ocular, rectal, long acting implantation,sublingual administration, under the tongue, from oral mucosa bypassingthe portal circulation, inhalation, or intranasal.

According to certain embodiments of the invention, a compositioncomprises an immunogenically effective amount of purified or partiallypurified adenovirus vector, such as an adenovirus 26 vector, comprisinga nucleic acid encoding a synthetic HIV envelope protein of theinvention. Said compositions can be formulated as a vaccine (alsoreferred to as an “immunogenic composition”) according to methods wellknown in the art.

Compositions of the invention can further optionally comprise anadjuvant to enhance immune responses. The terms “adjuvant” and “immunestimulant” are used interchangeably herein, and are defined as one ormore substances that cause stimulation of the immune system. In thiscontext, an adjuvant is used to enhance an immune response to thevectors encoding synthetic HIV envelope proteins of the invention and/orHIV antigenic polypeptides used in combination with vectors encodingsynthetic HIV envelope proteins of the invention.

Adjuvants suitable for use with the invention should be ones that arepotentially safe, well tolerated and effective in people, such as forinstance QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU,TERamide, PSC97B, Adjumer, PG-026, GSK-1, GcMAF, B-alethine, MPC-026,Adjuvax, CpG ODN, Betafectin, aluminum salts (e.g. AdjuPhos), Adjuplex,and MF59. The optimal ratios of each component in the formulation can bedetermined by techniques well known to those skilled in the art in viewof the present disclosure.

In a preferred embodiment, the adjuvant is an aluminum salt, such asAdjuPhos.

The preparation and use of immunogenic compositions are well known tothose of ordinary skill in the art. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol can also beincluded.

For instance recombinant adenovirus vector may be stored in the bufferthat is also used for the Adenovirus World Standard (Hoganson et al.,2002, Bioprocessing J 1: 43-8): 20 mM Tris pH 8, 25 mM NaCl, 2.5%glycerol. Another useful adenovirus formulation buffer suitable foradministration to humans is 20 mM Tris, 2 mM MgCl₂, 25 mM NaCl, sucrose10% w/v, polysorbate-80 0.02% w/v. Another formulation buffer that issuitable for recombinant adenovirus comprises 10-25 mM citrate buffer pH5.9-6.2, 4-6% (w/w) hydroxypropyl-beta-cyclodextrin (HBCD), 70-100 mMNaCl, 0.018-0.035% (w/w) polysorbate-80, and optionally 0.3-0.45% (w/w)ethanol. Obviously, many other buffers can be used, and several examplesof suitable formulations for the storage and for pharmaceuticaladministration of purified vectors are known.

According to embodiments of the invention, a composition of theinvention can be used together with one or more additional vectorsencoding one or more additional HIV antigenic polypeptides, and/or oneor more isolated HIV antigenic polypeptides. The additional vectorsand/or HIV antigenic polypeptides can be present in the same compositioncomprising a synthetic HIV Env protein of the invention. They can alsobe present in one or more different compositions that can be usedtogether with a composition comprising a synthetic HIV Env protein ofthe invention in a vaccine combination. Preferably, the one or moreadditional vectors are viral vectors, such as adenovirus vectors, andare most preferably adenovirus 26 vectors. The one or more additionalvectors can encode any HIV antigenic polypeptide known to those skilledin the art in view of the present disclosure.

In one embodiment, a composition or a vaccine combination furthercomprises a second adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigenic polypeptide comprising the amino acidsequence of SEQ ID NO: 5. An advantage of such embodiments is increasedbreadth of the immune response (covering strains from Clades B and C).

In another embodiment, a composition or a vaccine combination of theinvention further comprises an adenovirus vector, preferably anadenovirus 26 vector, encoding an HIV antigenic polypeptide comprisingthe amino acid sequence of SEQ ID NO: 28 (mos1GagPol).

In another embodiment, a composition or a vaccine combination of theinvention further comprises an adenovirus vector, preferably anadenovirus 26 vector, encoding an HIV antigenic polypeptide comprisingthe amino acid sequence of SEQ ID NO: 29 (mos2GagPol).

In a particular embodiment, a composition or a vaccine combination ofthe invention further comprises a second adenovirus vector, preferablyan adenovirus 26 vector, encoding a HIV antigenic polypeptide comprisingthe amino acid sequence of SEQ ID NO: 5, and one or more additionaladenovirus vectors, preferably adenovirus 26 vectors, encoding one ormore HIV antigenic polypeptides comprising the amino acid sequenceselected from the group consisting of SEQ ID NO: 28 or SEQ ID NO: 29.For example, a composition or a vaccine combination according to anembodiment of the invention can comprise four adenovirus vectors,preferably adenovirus 26 vectors, with a first vector encoding asynthetic HIV envelope protein comprising the amino acid sequence of SEQID NO: 8 (e.g. SEQ ID NO: 18); a second vector encoding a HIV antigenicpolypeptide comprising the amino acid sequence of SEQ ID NO: 5; a thirdvector encoding a synthetic HIV envelope protein comprising the aminoacid sequence of SEQ ID NO: 28; and a fourth vector encoding a syntheticHIV envelope protein comprising the amino acid sequence of SEQ ID NO:29.

In some embodiments, the composition or a vaccine combination furthercomprises one or more isolated HIV antigenic polypeptides. Any HIVantigenic polypeptide known to those skilled in the art in view of thepresent disclosure can be further included in a composition or a vaccinecombination of the invention, including, but not limited to an HIVenvelope protein (e.g., gp160, gp140, gp120, or gp4), preferably astabilized trimeric gp140 protein, such as a stabilized clade C or cladeA gp140 protein. In a preferred embodiment, the isolated HIV antigenicpolypeptide is a stabilized HIV clade C trimeric gp140 protein, such asthat comprising residues 30-708 of the amino acid sequence of SEQ IDNO:7 (residues 1-29 of SEQ ID NO:7 are in the signal sequence). Analternative or additional HIV Env polypeptide that could be used inaddition to the clade C gp140 protein or alone, is a mosaic Env trimerprotein, for instance having an amino acid sequence as disclosed inamino acids 30-724 of SEQ ID NO: 36 (corresponding to SEQ ID NO: 2 of WO2014/107744, residues 1-29 of SEQ ID NO:36 are in the signal sequence).

According to a particular embodiment of the invention, an HIV antigenicprotein can be a synthetic HIV envelope protein of the invention. Thus,a synthetic envelope protein of the invention can be used in isolatedand/or purified form to induce an immune response or provide aprotective immunity, etc. against HIV in a subject in need thereof. Anyof the synthetic envelope proteins described herein comprising the aminoacid sequence of SEQ ID NO: 8 can be used as an HIV antigenic protein inisolated and/or purified form. In a preferred embodiment, when used inisolated form as an HIV antigenic protein, the synthetic envelopeprotein comprises residues 30-711 of the amino acid sequence of SEQ IDNO: 18 or residues 30-686 of the amino acid sequence of SEQ ID NO: 19,and more preferably residues 30-704 of the amino acid sequence of SEQ IDNO: 18. The isolated HIV antigenic polypeptide can also comprise SEQ IDNO: 8 with the following mutations: EK479-480RRRR, I529P, A471C andT575C.

Embodiments of the invention also relate to compositions or vaccinecombinations comprising an isolated synthetic HIV envelope proteincomprising the amino acid sequence of SEQ ID NO: 8. Any of the syntheticHIV envelope proteins described herein can be used. In particularembodiments of the invention, the isolated synthetic HIV envelopeprotein comprises residues 30-704 or 30-711 of the amino acid sequenceof SEQ ID NO: 18, residues 30-686 of the amino acid sequence of SEQ IDNO: 19, or the amino acid sequence of SEQ ID NO: 8 with the followingmutations: EK479-480RRRR, I529P, A471C and T575C. Such compositions orvaccine combinations can further comprise one or more expressionvectors, e.g., adenoviral vectors such as adenovirus 26 vectors,encoding one or more additional HIV antigenic polypeptides, such as thesynthetic HIV envelope proteins of the invention, or other HIV antigenicproteins such as those set forth in SEQ ID NOs: 4, 5, 7, 28 or 29, orfragments thereof.

The invention also relates to a method of producing a composition or avaccine combination of the invention. According to embodiments of theinvention, a method of producing a composition or a combinationcomprises combining a vector comprising nucleic acid encoding thesynthetic HIV envelope protein of the invention with a carrier, andoptionally one or more additional vectors encoding one or moreadditional HIV antigenic polypeptides and/or one or more isolated HIVantigenic polypeptides. One of ordinary skill in the art will befamiliar with conventional techniques used to prepare such compositions.

Vaccine and Vaccine Combinations

According to embodiments of the invention, a composition can be avaccine. As used herein, the term “vaccine” refers to a compositioncomprising an expression vector, preferably a viral vector, encoding asynthetic HIV envelope protein of the invention that can provideprotective immunity or a protective immune response to a subject, or tovaccinate a subject. According to embodiments of the invention, uponadministration of the composition to a subject, the expression vectorexpresses the encoded synthetic HIV envelope protein, and the expressedsynthetic HIV envelope protein is presented to the immune system of thesubject, thereby inducing the required response to produce immunity, orinduce an immune response.

Thus, in another general aspect, the invention provides a vaccine forinducing an immune response against a human immunodeficiency virus (HIV)in a subject. According to embodiments of the invention, the vaccinecomprises a composition comprising an immunogenically effective amountof an expression vector encoding a synthetic HIV envelope proteincomprising the amino acid sequence of SEQ ID NO: 8, and preferably theamino acid sequence of SEQ ID NO:18. Preferably, the expression vectoris a viral vector, more preferably an adenovirus vector, and mostpreferably an adenovirus 26 vector.

According to embodiments of the invention, “inducing an immune response”when used with reference to the methods and compositions describedherein encompasses providing protective immunity and/or vaccinating asubject against an infection, such as a HIV infection, for prophylacticpurposes, as well as causing a desired immune response or effect in asubject in need thereof against an infection, such as a HIV infection,for therapeutic purposes. Preferably, the methods of the invention arefor prophylactic purposes, such as for providing protective immunity.The immune response can be a cellular immune response and/or a humoralimmune response.

As used herein, the term “protective immunity” or “protective immuneresponse” means that the vaccinated subject is able to control aninfection with the pathogenic agent against which the vaccination wasdone. Usually, the subject having developed a “protective immuneresponse” develops only mild to moderate clinical symptoms or nosymptoms at all. Usually, a subject having a “protective immuneresponse” or “protective immunity” against a certain agent will not dieas a result of the infection with said agent.

According to embodiments of the invention, vaccine compositions canfurther comprise one or more additional vectors, e.g., viral vectors,such as adenovirus vectors, preferably adenovirus 26 vectors, encodingone or more additional HIV antigenic polypeptides and/or one or moreisolated HIV antigenic polypeptides. The synthetic HIV envelope protein,additional vectors and/or one or more isolated HIV antigenicpolypeptides can be formulated in the same composition or one or moredifferent compositions in the vaccine.

The invention also relates to vaccine combinations for priming andboosting an immune response to one or more HIV clades in a subject inneed thereof using one or more vectors in combination with an isolatedantigenic polypeptide. Thus, in another general aspect, the inventionprovides a vaccine combination for inducing an immune response against aHIV in a subject. According to embodiments of the invention, the vaccinecombination comprises:

-   -   (i) a first composition comprising an immunogenically effective        amount of an expression vector encoding a synthetic HIV envelope        protein comprising the amino acid sequence of SEQ ID NO: 8 or        SEQ ID NO: 8 having one or more mutations selected from the        group consisting of (a) I529P, (b) K480E, and (c) a combination        of EK479-480RRRR, I529P, A471C and T575C, and a carrier; and    -   (ii) a second composition comprising an immunogenically        effective amount of an isolated HIV antigenic polypeptide and a        carrier,        wherein one of the first and second compositions is for priming        immunization and the other composition is for boosting        immunization.

According to embodiments of the invention, the vaccine combinationoptionally further comprises an immunogenically effective amount of oneor more additional expression vectors encoding one or more additionalHIV antigenic polypeptides. The one or more additional expressionvectors can be included in the first composition or the secondcomposition, or the one or more additional expression vectors can beincluded in one or more additional compositions to be administeredtogether with the first and/or second composition.

As used herein, the terms “co-delivery”, “co-administration” or“administered together with” refers to simultaneous administration oftwo or more components, such as a viral expression vector and anisolated antigenic polypeptide, or multiple viral expression vectors.“Simultaneous administration” can be administration of the two or morecomponents at least within the same day. When two components are“administered together with,” they can be administered in separatecompositions sequentially within a short time period, such as 24, 20,16, 12, 8 or 4 hours, or within 1 hour or less, or they can beadministered in a single composition at the same time.

In particular embodiments of a vaccine combination of the invention, thefirst composition comprises an adenovirus vector, preferably anadenovirus 26 vector, encoding a synthetic HIV envelope proteincomprising the amino acid sequence of SEQ ID NO: 18; and the isolatedHIV antigenic polypeptide comprises residues 30-708 of the amino acidsequence of SEQ ID NO: 7 or residues 30-724 of SEQ ID NO: 36. In oneparticular embodiment, the first composition further comprises anadenovirus vector, preferably an adenovirus 26 vector, encoding a HIVantigenic polypeptide comprising the amino acid sequence of SEQ ID NO:5. In another particular embodiment, the first composition furthercomprises one or more additional adenovirus vectors, preferablyadenovirus 26 vectors, encoding one or more additional HIV antigenicpolypeptides comprising the amino acid sequences selected from the groupconsisting of SEQ ID NOs: 28 and 29.

Another general aspect of the invention relates to a kit comprising avaccine combination according to an embodiment of the invention.

Other embodiments of the synthetic HIV envelope protein, expressionvectors, additional expression vectors, HIV antigenic polypeptidesencoded by the expression vectors, and isolated HIV antigenicpolypeptide etc. that can be used in the vaccine combinations of theinvention are discussed in detail above and in the illustrative examplesbelow.

Method for Inducing Protective Immunity Against HIV Infection

The invention also relates to a method of inducing an immune responseagainst one or more HIV clades in a subject in need thereof. The methodsdescribed herein include methods of priming and boosting an immuneresponse using one or more expression vectors in combination with one ormore isolated antigenic polypeptides.

In one general aspect, a method of inducing an immune response against ahuman immunodeficiency virus (HIV) in a subject comprises administeringto the subject a composition comprising an immunogenically effectiveamount of an expression vector comprising a nucleic acid encoding asynthetic HIV envelope protein comprising the amino acid sequence of SEQID NO: 8. Any of the compositions described herein can be used in amethod of inducing an immune response against HIV in a subject.Preferably, the composition comprises an adenovirus vector, preferablyan adenovirus 26 vector, comprising a nucleic acid encoding a syntheticHIV envelope protein comprising the amino acid sequence of SEQ ID NO:18. The composition can further comprise one or more additional vectorsencoding one or more additional HIV antigenic polypeptides and/or one ormore additional isolated HIV antigenic polypeptides.

In another general aspect, a method of inducing an immune responseagainst a human immunodeficiency virus (HIV) in a subject comprises:

-   -   (i) administering to the subject a first composition comprising        an immunogenically effective amount of an expression vector        encoding a mosaic HIV envelope protein having the amino acid        sequence of SEQ ID NO: 8 or SEQ ID NO: 8 having one or more        mutations selected from the group consisting of (a) I529P, (b)        K480E, and (c) a combination of EK479-480RRRR, I529P, A471C and        T575C, and a carrier;    -   (ii) administering to the subject a second composition        comprising an immunogenically effective amount of an isolated        HIV antigenic polypeptide and a carrier; and    -   (iii) optionally, administering to the subject an        immunogenically effective amount of one or more additional        expression vectors encoding one or more additional HIV antigenic        polypeptides,        wherein steps (i) and (ii) are conducted in either order, with        one of the steps for priming immunization and the other step for        boosting immunization. According to embodiments of the        invention, the optional, effective amount of the one more        additional expression vectors is administered together with the        first composition or the second composition. In a preferred        embodiment, the optional effective amount of the one or more        additional expression vectors is administered together with the        first composition.

In a particular embodiment of a method of inducing an immune response,the first composition comprises an adenovirus vector, preferably anadenovirus 26 vector, encoding a synthetic HIV envelope proteincomprising the amino acid sequence of SEQ ID NO: 8 and a secondadenovirus vector, preferably an adenovirus 26 vector, encoding a HIVantigenic polypeptide comprising the amino acid sequence of SEQ ID NO:5; the second composition comprises an isolated HIV antigenicpolypeptide having residues 30-708 of the amino acid sequence of SEQ IDNO:7 or residues 30-724 of SEQ ID NO:36; and the one or more additionalexpression vectors are adenovirus vectors, preferably adenovirus 26vectors, encoding one or more additional HIV antigenic polypeptidescomprising the amino acid sequences selected from the group consistingof SEQ ID NOs: 28 and 29; wherein the first composition is administeredto the subject, together with the one or more additional expressionvectors, one or more times for priming immunization, and the secondcomposition is administered to the subject one or more times forboosting immunization.

Administration of the immunogenic compositions comprising the expressionvectors and/or antigenic polypeptides is typically intramuscular,intradermal or subcutaneous. However, other modes of administration suchas intravenous, rectal, cutaneous, oral, nasal, etc can be envisaged aswell. Intramuscular administration of the immunogenic compositions canbe achieved by using a needle to inject a suspension of the expressionvectors, e.g. adenovirus vectors, and/or antigenic polypeptides. Analternative is the use of a needleless injection device to administerthe composition (using, e.g., Biojector™) or a freeze-dried powdercontaining the vaccine.

For intramuscular, intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the vector will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Likewise, the isolated antigenicpolypeptide will be in the form of a parenterally acceptable solutionhaving a suitable pH, isotonicity, and stability. Those of ordinaryskill in the art are well able to prepare suitable solutions using, forexample, isotonic vehicles such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives can be included, asrequired. A slow-release formulation can also be employed.

Typically, administration of the vaccine compositions according toembodiments of the invention will have a prophylactic aim to generate animmune response against an HIV antigen before infection or developmentof symptoms. In other embodiments, the expression vectors, e.g.,adenovirus vectors, and/or HIV antigenic polypeptides can beadministered for post-exposure prophylactics.

The immunogenic compositions containing the expression vectors, e.g.,adenovirus vectors, and/or antigenic polypeptides are administered to asubject, giving rise to an anti-HIV immune response in the subject. Anamount of a composition sufficient to induce a detectable immuneresponse is defined to be an “immunogenically effective dose” or“immunogenically effective amount.” In a typical embodiment of theinvention, the immune response is a protective immune response.

The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Prescription of treatment, e.g., decisions on dosage etc., iswithin the responsibility of general practitioners and other medicaldoctors, or in a veterinary context a veterinarian, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,16th edition, Osol, A. ed., 1980.

Following production of adenovirus vectors and optional formulation ofsuch particles into compositions, the vectors can be administered to anindividual, particularly a human or other primate. Delivery to anon-human mammal need not be for a therapeutic purpose, but can be foruse in an experimental context, for instance in investigation ofmechanisms of immune responses to the synthetic HIV envelope proteinexpressed by the adenovirus vectors of the invention.

In one embodiment of the disclosed methods, one or more adenovirusvectors encoding one or more HIV antigenic polypeptides are used toprime the immune response. One or more isolated HIV antigenicpolypeptides can be used together with the one or more adenovirusvectors for the priming immunization. The priming immunization may beadministered only once but can optionally also be administered multipletimes, for example, initial priming administration at time 0, followedby another priming administration about 4-14 weeks, e.g. 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 weeks, or any time in between, after the initialpriming administration. One or more isolated HIV antigenic polypeptidesoptionally together with one or more additional adenovirus or othervectors encoding one or more additional HIV antigenic polypeptides canbe used to boost the immune response. A boosting immunization can alsobe administered once or multiple times, for example, first at about18-36, e.g. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 or 36 weeks, or any time in between, after the initialpriming administration, followed by another boosting administration atabout 36-52, e.g. 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 weeks, or any time inbetween, after the initial priming administration. The immune responseinduced by the immunization is monitored.

Embodiments of the disclosed methods also contemplate shorterprime-boost regimens, meaning that the final boosting immunization isadministered about 22-26 weeks after the initial priming administration.The priming immunization can be administered at week 0. The boostingimmunization can be administered multiple times, for example, first atabout 7-9 weeks or 11-13 weeks, or at about 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14 weeks, or any time in between, after the initial primingadministration, followed by another boosting administration at about22-26 weeks, or at about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,or 28 weeks, or any time in between, after the initial primingadministration. In certain embodiments, one or more isolated HIVantigenic polypeptides is administered together with the one or moreadenovirus vectors for the priming and/or boosting immunization.

It is readily appreciated by those skilled in the art that the regimenfor the priming and boosting administrations can be adjusted based onthe measured immune responses after the administrations. For example,the boosting compositions are generally administered weeks or monthsafter administration of the priming composition, for example, about 2-3weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or28 weeks, or 30 weeks or 32 weeks or one to two years afteradministration of the priming composition.

According to embodiments of the invention, an adjuvant can beadministered together with the isolated HIV antigenic polypeptide aspart of the priming and/or boosting immunization. Any adjuvant can beused in view of the present disclosure, and in certain embodiments theadjuvant is an aluminum salt, such as AdjuPhos.

In a preferred embodiment of the invention, the adenovirus vectors usedin the methods disclosed herein include a rAd26 vector. Preferably, anrAd26 vector encoding a synthetic HIV envelope protein comprising theamino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, most preferablySEQ ID NO: 18, is used to prime the immune response, alone or incombination with one or more additional rAd26 vectors encoding one ormore additional HIV antigenic polypeptides, such as mos1Env having theamino acid sequence of SEQ ID NO:5, and an isolated HIV antigenicpolypeptide, such as that comprising residues 30-708 of the amino acidsequence of SEQ ID NO: 7 or residues 30-724 of SEQ ID NO: 36, is used toboost the immune response, or vice versa.

In one exemplary embodiment, an rAd26 vector encoding a synthetic HIVenvelope protein comprising the amino acid sequence of SEQ ID NO: 18 isused to prime the immune response in combination with an rAd26 vectorencoding an HIV antigenic polypeptide having the amino acid sequence ofSEQ ID NO: 5. One or more additional rAd26 vectors encoding one or moreadditional HIV antigenic polypeptides having the amino acid sequencesselected from the group consisting SEQ ID NOs: 1-4, 28 and 29 can alsobe administered together with the other rAd26 vectors to prime theimmune response. The priming administration in certain embodiments isadministered twice before any boosting immunization is administered. Anisolated HIV antigenic polypeptide, such as that comprising residues30-708 of the amino acid sequence of SEQ ID NO: 7 (preferably), or thatcomprising residues 30-724 of the amino acid sequence of SEQ ID NO:36,or a combination of at least two of such isolated HIV antigenicpolypeptides, is then administered to boost the immune response, and ispreferably administered more than once. Preferably, an adjuvant isfurther administered with the isolated HIV antigenic polypeptide in theboosting immunization.

In a particular embodiment, the an immune response is primed byadministration of four HIV antigens encoded on adenoviral vectors,preferably rAd26 vectors, the four antigens that are encoded being: (i)a synthetic HIV envelope protein comprising the amino acid sequence ofSEQ ID NO: 18, (ii) polypeptide having the amino acid sequence of SEQ IDNO: 5, (iii) polypeptide having the amino acid sequence of SEQ ID NO:28, and (iv) polypeptide having the amino acid sequence of SEQ ID NO:29. Each of these four antigens can be encoded on a separate adenoviralvector, preferably a rAd26 vector, administered at a total dose of about1, 2, 3, 4, 5, 6, 7, 8, 9, or 10×10¹⁰ viral particles (vp), e.g. about5×10¹⁰ vp (for all vectors together). The vectors may be pre-mixed, e.g.in a 1:1:1:1 ratio. The priming administration may be repeated after theinitial priming administration, e.g. at 8, 9, 10, 11, 12, 13, 14, 15 or16 weeks after the initial priming administration. In this embodiment,an immune response is boosted by administration of the same adenoviralvector vaccine used for the priming administration together withisolated HIV Env gp140 protein, e.g. clade C gp140 protein (comprisingresidues 30-708 of the amino acid sequence of SEQ ID NO: 7), or mosaicgp140 protein (comprising residues 30-724 of the amino acid sequence ofSEQ ID NO:36), or clade C gp140 protein and mosaic gp140 protein, at atotal dose of about 50-300 Ng protein, e.g. 50, 100, 150, 200, 250, or300 microgram, or any amount in between, of clade C gp140 protein, ore.g. 50, 100, 150, 200, 250, or 300 microgram, or any amount in between,of mosaic gp140 protein, or e.g. 50, 100, 150, 200, 250, or 300microgram, or any amount in between, of a combination of clade C gp140protein and mosaic gp140 protein (e.g. in a 1:1 ratio, either mixedtogether or separately administered). Preferably the gp140 protein isadministered together with adjuvant, e.g. aluminium phosphate. Theadenovirus plus gp140 protein administration to boost the immuneresponse may be performed at about 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30 weeks, or at any time in between, after the initialpriming administration. The boost administration may be repeated, e.g.at about 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 or 54 weeks, orany time in between, after the initial priming administration. Alladministrations according to this embodiment are preferably performedvia the intramuscular route.

Embodiments

Embodiment 1 is a nucleic acid encoding a synthetic HIV envelope proteincomprising the amino acid sequence of SEQ ID NO: 8, or SEQ ID NO: 8having one or more mutations selected from the group consisting of (i)I529P, (ii) K480E, and (iii) a combination of EK479-480RRRR, I529P,A471C and T575C.

Embodiment 2 is a nucleic acid according to embodiment 1, wherein thesynthetic HIV envelope protein further comprises a signal sequence, forinstance a signal sequence comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOs:9 to 12, preferably SEQ ID NO:9.

Embodiment 3 is a nucleic acid according to embodiment 1 or 2, whereinthe synthetic HIV envelope protein further comprises a transmembranedomain, for instance a transmembrane domain having the amino acidsequence of SEQ ID NO: 13, preferably the synthetic HIV envelope proteinfurther comprises SEQ ID NO:37 that is fused to the C-terminus of SEQ IDNO:8 and the N-terminus of the transmembrane domain.

Embodiment 4 is a nucleic acid according to embodiment 3, wherein thesynthetic HIV envelope protein further comprises a fragment of acytoplasmic domain, preferably a fragment of a cytoplasmic domaincomprising the amino acid sequence of SEQ ID NO: 14, or amino acidresidues 1-4 thereof (i.e., NRVR).

Embodiment 5 is a nucleic acid of any one of the preceding embodiments1-4, wherein the synthetic HIV envelope protein comprises the amino acidsequence of SEQ ID NO: 18.

Embodiment 6 is a nucleic acid according to embodiment 1 or 2, whereinthe synthetic HIV envelope protein either (a) further comprises atrimerization domain selected from the group consisting of GCN4,fibritin (foldon domain), for instance a trimerization domain having theamino acid sequence of SEQ ID NO: 15 or SEQ ID NO:16, preferably SEQ IDNO: 15, or (b) comprises SEQ ID NO:8 with a combination of the followingmutations: EK479-480RRRR, I529P, A471C and T575C.

Embodiment 7 is a nucleic acid according to embodiment 6, wherein thesynthetic HIV envelope protein comprises the amino acid sequence of SEQID NO: 19.

Embodiment 8 is a nucleic acid according to embodiment 5, wherein thesynthetic HIV envelope protein consists of the amino acid sequence ofSEQ ID NO: 18.

Embodiment 9 is a nucleic acid according to embodiment 7, wherein thesynthetic HIV envelope protein consists of the amino acid sequence ofSEQ ID NO: 19.

Embodiment 10 is a vector comprising the nucleic acid of any one ofembodiments 1-9, wherein the nucleic acid is operably linked to apromoter sequence.

Embodiment 11 is a vector according to embodiment 10 being a viralvector, preferably an adenovirus vector, and more preferably anadenovirus 26 vector.

Embodiment 12 is an isolated cell comprising the vector of embodiment 10or embodiment 11.

Embodiment 13 is a composition comprising an immunogenically effectiveamount of the vector of embodiment 10 or claim 11, and a carrier.

Embodiment 14 is a vaccine combination, comprising a first compositioncomprising an immunogenically effective amount of an adenovirus vector,preferably an adenovirus 26 vector, encoding a synthetic HIV envelopeprotein having the amino acid sequence of SEQ ID NO: 18, a secondcomposition comprising an immunogenically effective amount of a secondadenovirus vector, preferably a second adenovirus 26 vector, encoding anHIV antigenic polypeptide comprising the amino acid sequence of SEQ IDNO: 5, and optionally at least one additional composition comprising animmunogenically effective amount of at least one selected from the groupconsisting of a vector encoding an antigenic polypeptide having theamino acid sequence selected from the group consisting of SEQ ID NOs:1-4, 28 and 29, and an isolated HIV antigenic polypeptide havingresidues 30-708 of the amino acid sequence of SEQ ID NO: 7, or residues30-724 of the amino acid sequence of SEQ ID NO: 36, wherein the firstcomposition, second composition and additional composition are presentin the same composition or in one or more different compositions.

Embodiment 15 is a method of inducing an immune response against a humanimmunodeficiency virus (HIV) in a subject in need thereof, the methodcomprising administering to the subject the composition of embodiment 13or the vaccine combination of embodiment 14.

Embodiment 16 is a composition of embodiment 13 or a vaccine combinationof embodiment 14, comprising an adenovirus vector, preferably anadenovirus 26 vector, encoding a synthetic HIV envelope proteincomprising the amino acid sequence of SEQ ID NO: 18, a second adenovirusvector, preferably an adenovirus 26 vector, encoding an HIV antigenicpolypeptide comprising the amino acid sequence of SEQ ID NO: 5, one ormore additional adenovirus vectors encoding one or more additionalantigenic polypeptides comprising the amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-4, 28 and 29, and an isolated HIVantigenic polypeptide comprising residues 30-708 of the amino acidsequence of SEQ ID NO: 7 or residues 30-724 of SEQ ID NO: 36, for use ininducing an immune response against a human immunodeficiency virus(HIV).

Embodiment 17 is a synthetic HIV envelope protein comprising the aminoacid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 having one or moremutations selected from the group consisting of (i) I529P, (ii) K480E,and (iii) a combination of EK479-480RRRR, I529P, A471C and T575C.

Embodiment 18 is a synthetic HIV envelope protein of embodiment 17,comprising the amino acid sequence of SEQ ID NO: 8 with a combination ofmutations EK479-480RRRR, I529P, A471C and T575C, or residues 30-704 ofthe amino acid sequence SEQ ID NO: 18 or residues 30-686 of SEQ ID NO:19.

Embodiment 19 is a composition of embodiment 13, further comprising oneor more additional expression vectors encoding one or more additionalHIV antigenic polypeptides, and/or one or more isolated HIV antigenicpolypeptides.

Embodiment 20 is a composition of embodiment 13 comprising an adenovirusvector, preferably an adenovirus 26 vector, encoding a synthetic HIVenvelope protein consisting of the amino acid sequence of SEQ ID NO: 18.

Embodiment 21 is a composition according to embodiment 20 furthercomprising a second adenovirus vector, preferably an adenovirus 26vector, encoding an HIV antigenic polypeptide comprising the amino acidsequence of SEQ ID NO: 5, and optionally one or more additionaladenovirus vectors, preferably adenovirus 26 vectors, encoding one ormore additional HIV antigenic polypeptides comprising the amino acidsequences of SEQ ID NOs: 1-4, 28 and 29.

Embodiment 22 is a method of producing an immune response against ahuman immunodeficiency virus (HIV) in a subject in need thereof, themethod comprising administering to the subject a composition accordingto any one of embodiments 19, 20, or 21.

Embodiment 23 is a method of producing a composition or a vaccinecombination, comprising combining the vector of embodiment 10 orembodiment 11 with a carrier, and optionally one or more additionalvectors encoding one or more additional HIV antigenic polypeptidesand/or one or more isolated HIV antigenic polypeptides in one or morecompositions, together with a carrier.

Embodiment 24 is a vaccine combination for inducing an immune responseagainst a human immunodeficiency virus (HIV) in a subject, comprising:

-   -   (i) a first composition comprising an immunogenically effective        amount of an expression vector encoding a synthetic HIV envelope        protein comprising the amino acid sequence of SEQ ID NO: 8 or        SEQ ID NO: 8 having one or more mutations selected from the        group consisting of (i) I529P, (ii) K480E, and (iii) a        combination of EK479-480RRRR, I529P, A471C and T575C, and a        carrier; and    -   (ii) a second composition comprising an immunogenically        effective amount of an isolated HIV antigenic polypeptide and a        carrier,

wherein one of the first and second compositions is for primingimmunization and the other composition is for boosting immunization, and

wherein the vaccine combination optionally further comprises animmunogenically effective amount of one or more additional expressionvectors encoding one or more additional HIV antigenic polypeptides, andthe one or more additional expression vectors are included in the firstor the second composition or one or more additional compositions to beused together with the first or second composition.

Embodiment 25 is a vaccine combination according to embodiment 24,wherein the first composition comprises an adenovirus vector, preferablyan adenovirus 26 vector, encoding a synthetic HIV envelope proteincomprising the amino acid sequence of SEQ ID NO: 18; the isolated HIVantigenic polypeptide comprises residues 30-708 of the amino acidsequence of SEQ ID NO: 7, or residues 30-724 of SEQ ID NO: 36; and theone or more additional expression vectors are adenovirus vectors,preferably adenovirus 26 vectors, encoding one or more additional HIVantigenic polypeptides comprising the amino acid sequences selected fromthe group consisting of SEQ ID NOs: 1-5, 28 and 29.

Embodiment 26 is a method of inducing an immune response against a humanimmunodeficiency virus (HIV) in a subject in need thereof, the methodcomprising:

-   -   (i) administering to the subject a first composition comprising        an immunogenically effective amount of an expression vector        encoding a synthetic HIV envelope protein comprising the amino        acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 having one or more        mutations selected from the group consisting of (i) I529P, (ii)        K480E, and (iii) a combination of EK479-480RRRR, I529P, A471C        and T575C, and a carrier;    -   (ii) administering to the subject a second composition        comprising an immunogenically effective amount of an isolated        HIV antigenic polypeptide and a carrier; and    -   (iii) optionally, administering to the subject an        immunogenically effective amount of one or more additional        expression vectors encoding one or more additional HIV antigenic        polypeptides,        wherein steps (i) and (ii) are conducted in either order, with        one of the steps for priming immunization and the other step for        boosting immunization, and preferably, the optional, effective        amount of the one more additional expression vectors is        administered together with the first composition.

Embodiment 27 is a method according to embodiment 26, wherein the firstcomposition comprises an adenovirus vector, preferably an adenovirus 26vector, encoding a synthetic HIV envelope protein having the amino acidsequence of SEQ ID NO: 18 and a second adenovirus vector, preferably andadenovirus 26 vector, encoding a HIV antigenic polypeptide comprisingthe amino acid sequence of SEQ ID NO: 5; the second compositioncomprises an isolated HIV antigenic polypeptide having residues 30-708of the amino acid sequence of SEQ ID NO: 7, or residues 30-724 of SEQ IDNO: 36; and the optional one or more additional expression vectors areadenovirus vectors, preferably adenovirus 26 vectors, encoding one ormore additional HIV antigenic polypeptides comprising the amino acidsequences selected from the group consisting of SEQ ID NOs. 28 and 29;wherein the first composition is administered to the subject, optionallytogether with the one or more additional expression vectors, one or moretimes for priming immunization, and the second composition isadministered to the subject one or more times for boosting immunization.

Embodiment 28 is a synthetic HIV envelope protein consisting of theamino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, with or withoutthe signal sequence.

Embodiment 29 is a vaccine combination comprising one or more vectorstogether comprising nucleic acid sequences encoding (i) a firstsynthetic HIV envelope protein comprising the amino acid sequence of SEQID NO: 8 and (ii) a second HIV envelope protein comprising the aminoacid sequence of SEQ ID NO: 5.

Embodiment 30 is a vaccine combination according to embodiment 29,wherein the first synthetic HIV envelope protein comprises the aminoacid sequence of SEQ ID NO: 18.

Embodiment 31 is a vaccine combination comprising the followingcomponents:

(i) an Ad26 vector encoding a synthetic HIV envelope protein consistingof the amino acid sequence of SEQ ID NO: 18; and(ii) an Ad26 vector encoding an HIV envelope protein consisting of theamino acid sequence of SEQ ID NO: 5.

Embodiment 32 is a vaccine combination according to embodiment 31,further comprising the following component:

(iii) an Ad26 vector encoding HIV antigens consisting of the amino acidsequence of SEQ ID NO: 28.

Embodiment 33 is a vaccine combination according to embodiment 31 or 32,further comprising the following component:

(iv) an Ad26 vector encoding HIV antigens consisting of the amino acidsequence of SEQ ID NO: 29.

Embodiment 34 is a vaccine combination according to any one ofembodiments 31-33, further comprising the following component:

(v) isolated HIV antigenic polypeptide having residues 30-708 of theamino acid sequence of SEQ ID NO: 7, or residues 30-724 of the aminoacid sequence of SEQ ID NO: 36, optionally further comprising anadjuvant.

Embodiment 35 is a method of inducing an immune response against a humanimmunodeficiency virus (HIV) in a human subject in need thereof, themethod comprising:

(a) administering to the subject: (i) a rAd26 vector encoding asynthetic HIV envelope protein comprising the amino acid sequence of SEQID NO: 18; (ii) a rAd26 vector encoding an antigen comprising the aminoacid sequence of SEQ ID NO: 5; (iii) a rAd26 vector encoding an antigencomprising the amino acid sequence of SEQ ID NO: 28; and (iv) a rAd26vector encoding an antigen comprising the amino acid sequence of SEQ IDNO: 29; preferably wherein the rAd26 vectors are administered in a ratioof about 1:1:1:1 at a total dose of about 1-10×10¹⁰ viral particles(vp), e.g. 5×10¹⁰ vp;(b) repeating step (a) at about 10-14 weeks, e.g. at 12 weeks after step(a);(c) administering to the subject: (i) a rAd26 vector encoding asynthetic HIV envelope protein comprising the amino acid sequence of SEQID NO: 18; (ii) a rAd26 vector encoding an antigen comprising the aminoacid sequence of SEQ ID NO: 5; (iii) a rAd26 vector encoding an antigencomprising the amino acid sequence of SEQ ID NO: 28; (iv) a rAd26 vectorencoding an antigen comprising the amino acid sequence of SEQ ID NO: 29;(v) isolated HIV gp140 protein having the sequence of amino acids 30-708of SEQ ID NO: 7; and (vi) aluminium phosphate adjuvant; preferablywherein the rAd26 vectors are administered in a ratio of about 1:1:1:1at a total dose of about 1-10×10¹⁰ viral particles (vp), e.g. 5×10¹⁰ vpand wherein the isolated HIV gp140 protein is administered at a dose ofabout 50-300 microgram, e.g. 250 microgram; at about 20-28 weeks, e.g.at 24 weeks after step (a); and (d) repeating step (c) at about 42-54weeks, e.g. at 48 weeks after step (a).

Embodiment 36 is a method of inducing an immune response against a humanimmunodeficiency virus (HIV) in a human subject in need thereof, themethod comprising:

(a) administering to the subject: (i) a rAd26 vector encoding asynthetic HIV envelope protein comprising the amino acid sequence of SEQID NO: 18; (ii) a rAd26 vector encoding an antigen comprising the aminoacid sequence of SEQ ID NO: 5; (iii) a rAd26 vector encoding an antigencomprising the amino acid sequence of SEQ ID NO: 28; and (iv) a rAd26vector encoding an antigen comprising the amino acid sequence of SEQ IDNO: 29; preferably wherein the rAd26 vectors are administered in a ratioof about 1:1:1:1 at a total dose of about 1-10¹⁰ viral particles (vp),e.g. 5×10¹⁰ vp;(b) repeating step (a) at about 10-14 weeks, e.g. at 12 weeks after step(a);(c) administering to the subject: (i) a rAd26 vector encoding asynthetic HIV envelope protein comprising the amino acid sequence of SEQID NO: 18; (ii) a rAd26 vector encoding an antigen comprising the aminoacid sequence of SEQ ID NO: 5; (iii) a rAd26 vector encoding an antigencomprising the amino acid sequence of SEQ ID NO: 28; (iv) a rAd26 vectorencoding an antigen comprising the amino acid sequence of SEQ ID NO: 29;(v) isolated HIV gp140 protein having the sequence of amino acids 30-708of SEQ TD NO: 7; (vi) isolated HIV gp140 protein having the sequence ofamino acids 30-724 of SEQ ID NO: 36; and (vii) aluminium phosphateadjuvant; preferably wherein the rAd26 vectors are administered in aratio of about 1:1:1:1 at a total dose of about 1-10×10¹⁰ viralparticles (vp), e.g. 5×10¹⁰ vp and wherein the isolated HIV gp140proteins are administered in a ratio of about 1:1 at a total dose ofabout 50-300 microgram, e.g. 250 microgram; at about 20-28 weeks, e.g.at 24 weeks after step (a); and(d) repeating step (c) at about 42-54 weeks, e.g. at 48 weeks after step(a).

EXAMPLES Example 1: Design of HIV Envelope Antigen Sequences

Several HIV envelope antigen sequences were designed having sequencesimilarity to the mosaic HIV antigen mos2Env (SEQ ID NO: 6; previouslyalso described in WO 2010/059732). The newly designed, membrane bound,sequences were based on (a combination of) fully natural wild-typesequences from HIV envelope proteins, or a chimera of mos2Env sequenceand wild-type HIV envelope protein sequences. In addition to full lengthenvelope protein sequences (see FIG. 1A), sequences having a C-terminaltruncation of the cytoplasmic domain were also designed (see, e.g., FIG.1C). See also e.g., Schiernle et al., PNAS 1997; Abrahamyan et al., JVirol 2005); Edwards et al., J. Virology, 2002, 76:2683-2691. Solublevariants were also prepared by C-terminal truncation before thetransmembrane (TM) region, which was replaced by a trimerization domain,such as a GCN4 trimerization domain (see, e.g., FIG. 1B). These solublevariants were further converted into a single chain variant by mutationof the furin-cleavage site, thus inhibiting the processing of theextracellular domain of the envelope protein into gp120 and gp41subunits.

Of the all the constructs generated and tested, constructs based on C4had the most optimal properties, e.g., good manufacturability, folding,immunogenicity, etc. and these were selected for further studies. Asoluble variant of the C4 construct having a GCN4 trimerization domainin place of the transmembrane domain (sC4, FIG. 1B), and a variantcomprising a 7-amino acid fragment of the cytoplasmic domain (C4D7, FIG.1C) were also generated and tested in further studies. The amino acidsequences of C4, sC4, and C4D7 are shown in SEQ ID NOs: 17, 19, and 18,respectively. Sequences encoding these are shown in SEQ ID NOs: 25, 27,and 26, respectively. Construct C1 has an extracellular domain sequencebased on the mos2Env sequence (SEQ ID NO: 6). A soluble variant ofconstruct C1 having a GCN4 trimerization domain in place of thetransmembrane domain (sC1), and a variant comprising a 7-amino acidfragment of the cytoplasmic domain (C1D7), similar to sC4 and C4D7 asshown in FIGS. 1B and 1C, respectively, were also generated. ConstructC1 and its variants were used in further studies for comparisonpurposes, since these are essentially based on the mos2Env sequence ofthe prior art. The amino acid sequences of C1, sC1 and C1D7 are shown inSEQ ID NOs: 31, 30, and 32, respectively. Nucleic acid sequencesencoding these are shown in SEQ ID NOs: 34, 33, and 35, respectively.Other constructs that were tested were less optimal than the ones basedon construct C4, and were not taken into further development.

Example 2: Expression and Folding of Synthetic HIV Envelope Proteins

The expression level, folding, and cell-surface expression of syntheticHIV envelope proteins were measured.

Expression Levels

HEK293F cells were transiently transfected with a plasmid encoding thesoluble synthetic HIV envelope proteins sC1 and sC4 as described inExample 1. Expression levels of the soluble protein were measured in thesupernatant using quantitative Western blot (QWB). The results are shownin FIG. 2. The low expression levels for sC1 (which essentiallycorresponds to mos2Env with an added transmembrane domain) are in linewith our recent insights for mos2Env. As demonstrated by the results,the sC4 variant of the invention showed significantly higher expressionlevels than the sC1 variant (control).

Protein Folding

Protein folding was tested by measuring the binding of soluble syntheticHIV envelope proteins to an antibody (MAb 17b) known to bind theco-receptor binding site of the HIV envelope protein, which is exposedonly after binding of CD4, by enzyme-linked immunosorbent assay (ELISA).In particular, binding of purified sC4 was tested for binding to MAb 17bwith prior binding of sC4 to CD4, and without prior binding of sC4 toCD4. Purified sC1 was used as a control. Binding of MAb 17b to sC4without prior CD4 binding to the envelope protein is an indication ofpartially unfolded or pre-triggered envelope protein (i.e., an unstableEnv that adopts the “open” conformation in the absence of CD4 binding).The results of the ELISA assay are shown in FIGS. 3A and 3B.

As shown in FIG. 3B, sC4 shows strong binding to MAb 17b with priorbinding to CD4, but no detectable binding to MAb 17b without priorbinding to CD4. In contrast, as shown in FIG. 3A, sC1 showed much lowerbinding to MAb 17 both with and without prior binding to CD4. Theresults suggest that sC4 has a correct folding pattern, with no exposureof the co-receptor binding site prior to CD4 binding.

Protein folding was also analyzed by native polyacrylamide gelelectrophoresis (PAGE) of sC1 and sC4 to evaluate the quaternarystructure of the soluble protein variants, and possible incorrectdisulfide bridge formation between protomers. After electrophoresis on anative gel, protein in the gel was detected by Western blot analysis. Asshown by the results in FIG. 4, the majority of sC4 is present in atrimeric state, which is the correct quaternary structure.

Taken together, the results of the protein folding experimentsdemonstrate that the sC4 soluble synthetic HIV envelope protein has thedesired folding profile, which is improved as compared to the foldingprofile of the existing mos2Env antigen (represented by sC1).

Cell Surface Expression

Cell surface expression of the membrane-bound variants of HIV envelopeproteins C1 (full length), C4 (full length, see FIG. 1A), C1D7, and C4D7was also studied. HEK293T cells were transiently transfected with onlyeGFP-encoding plasmid (negative control, NC), or with eGFP-encodingplasmid together with an expression construct encoding an HIV envelopeprotein variant. Two days post-transfection, cells were subjected tofluorescence activated cell sorting (FACS)-analysis upon exposure toseveral poly- and monoclonal antibodies directed against gp120, andsecondary antibodies, and then examined for envelope proteincell-surface expression levels. Quality of the envelope variants wasassessed by determining the overall expression levels using ananti-gp120 polyclonal antibody, and by assessing relative binding of thebroadly neutralizing antibodies PG9 and PG16, which arequaternary-structure dependent, and preferentially bind to correctlyfolded envelope trimer.

The results of the cell surface expression experiments are shown in FIG.5. The surface expression levels of truncated variants C1D7 and C4D7 asmeasured using an anti-gp1 20 antibody, are much higher than the surfaceexpression levels of their full length counterparts, C1 and C4,respectively. This confirms that deletion of 144 residues from thecarboxy-terminus of Env increases envelope surface expression levels.The full length C4 construct of the invention also showed improved PG9and PG16 binding as compared to full length C1, suggesting that the C4envelope sequence is properly folded (i.e., a trimer) on the cellsurface.

The results also demonstrate that the C1D7 variant, which is essentiallyMos2Env with an added transmembrane domain and 7 amino acids of thecytoplasmic domain, can be surface-expressed on HEK293T cells. This isin contrast to the soluble construct in Ad26.mos2Env, which cannot beexpressed at detectable levels on the surface when transfected to A549cells. However, relative binding to PG9 and PG16 is barely detectableabove background, suggesting that the C1D7 envelope sequence is poorlyfolded and is probably not present as an intact trimer on the cellsurface.

Overall, the C4D7 envelope variant has the most optimal antibody bindingprofile, with higher gp120 expression than its full-length counterpartC4, and with greater than 15-fold increased PG9 and PG16 bindingcompared to C1 and C1D7 (FIG. 5).

Example 3: Stability of Vectors Encoding HIV Envelope Sequences

Previous work in our laboratories (unpublished) indicated thatadenovirus 26 (Ad26) vectors encoding the mos2Env antigen sequenceshowed had relatively high VP/IU ratios (indicating lower quality ofadenovirus product batches) and moreover that such vectors displayedstability issues. Accordingly, it was important to test the stability ofthe synthetic HIV envelope proteins constructs of the invention in anadenovirus background.

Recombinant Ad26 (rAd26) vectors encoding HIV antigen sequences of theinvention C4, C4D7, and sC4 as described above in Example 1 weregenerated in PER.C6 cells (referred to as “rAd26.CA”, “rAd26.C4D7”, and“rAd26.sC4”, respectively). Vector clones (plaques) were picked andscaled-up for the generation of research batches. A maximum of 5 viralclones (plaques) were scaled-up to T25 format and serially passaged for10 passages in T25 format (passages 1-3 being the transfection andplaque purification steps, followed by 10 passages in T25 format,resulting in a total of 13 passages). Genetic stability was assessed atviral passage number (vpn) 3, 5, 10 and 13 by an E1 transgene cassettePCR assay, followed by sequencing at vpn 13. The results are shown inFIG. 6.

The rAd26 vectors encoding full length C4 (rAd26.C4) showed poor growthcharacteristics, as determined by no full cytopathogenic effect (CPE) in2-3 days; genetic instability, as determined by deletions of the E1transgene cassette region; or a combination thereof (FIG. 6). Due to thepoor growth characteristics and observed genetic instability, thisvector encoding full length C4 was not pursued further.

In contrast, for the rAd26 vectors encoding C4D7 (rAd26.C4D7) and sC4(rAd26.sC4), all propagated plaques remained genetically stable duringthe course of the experiment (FIG. 6). Thus, the novel sC4 and C4D7constructs outperform the original mos2Env construct with respect tostability in an adenoviral vector background. The genetic stabilitytesting up to vpn 13 represents propagation several passages beyond thatused in the industrial scale preparation of the vectors.

Example 4: Expression and In Vivo Antigenicity of HIV Envelope Sequencesin Adenovirus Vectors

Expression and antigenicity of rAd26.C4D7 and rAd26.sC4 were assessedseparately or in combination with a recombinant Ad26 vector encodingmos1Env (SEQ ID NO: 5) (hereinafter “rAd26.mos1Env”) invector-transduced A549 cells (human cell line) in vitro (data notshown). Flow cytometry analysis demonstrated that all antigens wereexpressed in cell cultures transduced with either 2×10⁴ viral particles(vp) of the single envelope antigens as controls, or with 1×10⁴ vp ofthe 2 combined Env antigens by adenovirus transduction. Alltransductions additionally contained single doses (1×10⁴ vp) ofadenovirus vectors encoding mos1GagPol (“rAd26.mos1GagPol”) andmos2GagPol (“rAd26.mos2GagPol”) (Barouch et al, Nat Med 2010,16:319-323), so that the assessed vector combinations exhibited the samerelative ratios of the different adenoviral vectors as intended forpre-clinical and clinical use. Preferably, the vectors encodingsynthetic HIV envelope proteins of the invention are combined withvectors encoding the mos1GagPol and the mos2GagPol antigens for clinicaluse.

The combination of rAd26.mos1Env and rAd26.C4D7 yielded a maximalcoverage of the assessed epitopes as determined by monoclonal antibodybinding. Particularly, the exposure of the PG16 epitope, which wascontributed by transformation with Ad26.C4D7 is promising for vaccineuse since PG16 represents a broadly neutralizing monoclonal antibodyrecognizing the V1/V2 loop region of HIV-1 Env (Walker et al, Science.2009). Hence, the synthetic HIV envelope protein of the inventionderived from the C4 sequence increases the breadth of the immuneresponse against the HIV envelope protein compared to the immuneresponse generated by mos1Env only. Vaccine-induced antibody responsesdirected towards the envelope protein region have been shown tocorrelate with protection from HIV-1 infection in the RV144 study(Haynes et al, N Engl J Med. 2012), and thus the synthetic HIV envelopeprotein of the invention is a promising candidate to include in HIVvaccine regimens.

Example 5: Immunogenicity of Vectors Encoding Synthetic HIV EnvelopeProteins

The synthetic HIV envelope protein sequences of the invention in an Ad26vector background were tested in rabbits to determine if theseconstructs were an immunogenic alternative to the rAd26.mos2Envconstruct.

The immunogenicity of adenovirus vector encoding mos1Env (rAd26.mos1Env;SEQ ID NO: 5) was tested alone, and in combination with adenovirusvectors encoding synthetic HIV envelope proteins of the invention(rAd26.C4D7 and rAd26.sC4; comprising SEQ ID NO: 8, in particular SEQ IDNOs: 18 and 19, respectively). In all cases, adenovirus 26 vectorsencoding mos1GagPol and mos2GagPol antigens (rAd26.mos1GagPol [SEQ IDNO: 28] and rAd26.mos2GagPol [SEQ ID NO: 29], respectively) were alsoadministered. More specifically, the immunogenicity of rAd26.mos1Envalone (trivalent vaccine: rAd26.mos1GagPol, rAd26.mos2GagPol andrAd26.mos1Env) was compared to the immunogenicity of rAd26.mos1Env incombination with one of rAd26.C4D7 or rAd26.sC4 (tetravalent vaccine:administration of either rAd26.mos1GagPol, rAd26.mos2GagPol,rAd26.mos1Env and rAd26.C4D7; or administration of rAd26.mos1GagPol,rAd26.mos2GagPol, rAd26.mos1Env and rAd26.sC4). This comparison of thetrivalent vaccine, which lacks any vectors encoding the synthetic HIVenvelope proteins of the invention, with the tetravalent vaccine, whichcontains vectors encoding the synthetic HIV envelope proteins of theinvention, allows for a determination of whether the HIV envelopeproteins of the invention contribute to the breadth of protection.

Administration was done in vaccine regimens, wherein these Ad26 vectorswere administered at weeks 0 and 6 as a double prime, and a clade Cgp140 protein (a trivalent Env gp140 protein having SEQ ID NO: 7 withoutthe signal peptide sequence of residues 1-29, see also WO 2010/042942)at weeks 12 and 18 as a double boost (see e.g. Barouch et al, 2015,Science 349: 320-324). Table 1 describes the vaccine regimens used forthe current study, rAd26. Empty refers to a control vector lacking anygene encoding a sequence for an HIV antigenic protein. Each groupcontained six rabbits.

TABLE 1 Vaccine regimens tested in immunogenicity study in rabbits Firstand second Immunizations Third and fourth immunizations Total dose DoseGroup adeno vectors Dose (vp) (vp) protein boost (ug) Adjuvant N = 1rAd26.Mos1Env  2.5 × 10¹⁰ 5 × 10¹⁰ GP140 (clade C) 10 AdjuPhos 250 μg 6rAd26.Mos1GagPol 1.25 × 10¹⁰ rAd26.Mos2Gagpol 1.25 × 10¹⁰ 2rAd26.Mos1Env 1.25 × 10¹⁰ 5 × 10¹⁰ GP140 (clade C) 10 AdjuPhos 250 μg 6rAd26.C4D7 1.25 × 10¹⁰ rAd26.Mos1GagPol 1.25 × 10¹⁰ rAd26.Mos2Gagpol1.25 × 10¹⁰ 3 rAd26.Mos1Env 1.25 × 10¹⁰ 5 × 10¹⁰ GP140 (clade C) 10AdjuPhos 250 μg 6 rAd26.sC4 1.25 × 10¹⁰ rAd26.Mos1GagPol 1.25 × 10¹⁰rAd26.Mos2Gagpol 1.25 × 10¹⁰ control rAd26.Empty   5 × 10¹⁰ 5 × 10¹⁰ NA0 AdjuPhos 250 μg 6

The comparison of the trivalent Ad26 vaccine (lacking the novel Envantigens of the invention) with the tetravalent Ad26 vaccine (whichcomprises the novel sC4 or C4D7 Env antigens) allows for testing if thenovel antigens contribute to breadth of protection. An establishedTZM-b1 cell-based neutralization assay [Montefiori D C. Methods Mol Biol2009, 485:395-405; Sarzotti-Kelsoe M et al., J Immunol Methods 2014,409:131-146] was used to measure neutralizing activity of the vaccinecandidates.

Results are shown in FIG. 7, and were statistically analyzed by usingthe trivalent vaccine (group 1 in Table 1) as control group andcomparing to each of the novel quadrivalent vaccines (groups 2 and 3 inTable 1).

Overall, the novel C4-derived (i.e. encoding Env proteins comprising SEQID NO: 8, being an alternative for mos2Env) adeno constructs wereimmunogenic after two homologous intramuscular immunizations in rabbits.

Neutralization capacity of rabbit immune sera against Tier 1Bpseudoviruses was absent (data not shown), which is not unexpected as itwas known that such viruses are more difficult to neutralize.

Pseudovirus neutralization capacity of rabbit immune sera against aclade B Tier 1A virus was unaffected by the addition of new components(data not shown). This demonstrates that the novel antigen did notnegatively interfere with immunogenicity of the existing clade B antigenpresent in the vaccine (although the new components were directed toclade C, such undesirable interference could not be excluded a prioribefore it had been tested).

Pseudovirus neutralization capacity of rabbit immune sera against aclade C Tier 1A virus was significantly enhanced in the quadrivalentnovel C4D7 containing adeno (quadrivalent, group 2), compared totrivalent (having only mos1Env) immunization alone (group 1) (FIG. 7panel B). In addition, pseudovirus neutralization capacity of rabbitimmune sera against a clade C Tier 1A virus at week 8 was significantlyenhanced in the quadrivalent novel sC4 containing adeno (quadrivalent,group 3), compared to trivalent (having only mos1Env) immunization alone(group 1) (FIG. 7 panel B).

In conclusion, the C4D7 and sC4 constructs encoded in Ad26 wereimmunogenic and addition thereof expanded the binding- andneutralization capacity of a vaccine that has mos1Env (mainly clade B)as sole Ad26-encoded Env component, towards clade C strains (FIG. 7B).

Example 6: Immunogenicity of Vaccine Regimens Including Vectors EncodingSynthetic HIV Envelope Proteins of the Invention

One further rabbit study assessed the tetravalent vector combinationAd26.Mos4.HIV (consisting of four adenoviral vectors: Ad26.Mos1GagPol[encoding SEQ ID NO: 28], Ad26.Mos2GagPol [encoding SEQ ID NO: 29],Ad26.Mos1Env [encoding SEQ ID NO: 5] and Ad26.Mos2SEnv [the name “C4D7”as used above is also referred to as “Mos2S”; this vector encodes thenovel SEQ ID NO: 18 according to the invention], in a 1:1:1:1 mixture ata total dose of 5×10⁹ vp,) applied intramuscularly as double primeimmunizations in weeks 0 and 6, in combination with recombinant HIV-1Env protein boosts using Clade C gp140 (having the sequence of aminoacid residues 30-708 of SEQ ID NO: 7), Mosaic gp140 (having the sequenceof amino acid residues 30-724 of SEQ ID NO: 361, or a combination ofClade C gp140 and Mosaic gp140, in weeks 13 and 19. These protein boostswere applied intramuscularly at a total dose of 10 or 50 micrograms ofprotein combined with 250 mcg aluminum phosphate adjuvant formulated onthe day of immunization.

Results indicate that all tested regimens were immunogenic in allanimals, inducing high antibody titers and moderate neutralizationactivity against Tier 1 Env pseudotyped viruses. If Mosaic gp140 wasused as vaccine antigen, either alone or in combination with Clade Cgp140. Mosaic gp140-specific ELISA titers and Clade B pseudovirusrecognition were significantly increased at week 15 in comparison to thereference group boosted with Clade C gp140 alone. The overall effectsize of the improvement was moderate, and bigger for the group boostedwith the bivalent Clade C gp140—Mosaic gp140 combination compared toMosaic gp140 alone. At week 21 of the study, these differences were lostand immune responses measured for the cohorts receiving bivalent Clade Cgp140—Mosaic gp140 boosts or monovalent Clade C gp140 boosts werestatistically indistinguishable.

The bivalent protein regimen showed comparable induction of Clade CELISA titers and pseudovirus recognition as the Clade C gp140 aloneboosted regimen, indicating that the inclusion of the clade B-relatedimmunogen Mosaic gp140 had no negative effect on clade C antigencoverage, whilst significantly enhancing clade B coverage at week 15 ofthe study.

The data confirm that the Ad26.Mos2SEnv vector encoding a synthetic Envantigen according to the invention can successfully used in vaccineregimens.

REFERENCES

-   1. Barouch et al, Nat Med 2010, 16: 319-323-   2. WO 2010/059732-   3. Schiernle et al., PNAS 94: 8640-8645, 1997-   4. Abrahamyan et al., J Virol 79: 106-115, 2005-   5. US20120076812-   6. Barouch et al., Cell 155:1-9, 2013-   7. Havenga, et al., 2006, J Gen Virol 87: 2135-43;-   8. WO 03/104467-   9. WO 2004/001032-   10. WO 2007/104792-   11. Abbink et al., (2007) Virol 81(9): 4654-63-   12. U.S. Pat. No. 7,270,811-   13. Vogels et al., (2003) J Virol 77(15): 8263-71-   14. WO 00/70071-   15. WO2012/082918-   16. Walker L M, Phogat S K, Chan-Hui P Y, Wagner D, Phung P. Goss J    L, et al. Broad and potent neutralizing antibodies from an African    donor reveal a new HIV-1 vaccine target. Science 2009, 326:285-289.-   17. Haynes B F, Gilbert P B, McElrath M J, Zolla-Pazner S, Tomaras G    D, Alam S M, et al. Immune-correlates analysis of an HIV-1 vaccine    efficacy trial. N Engl J Med 2012, 366:1275-1286.-   18. Barouch et al. (2015) Science 349: 320-324-   19. Montefiori D C. Measuring HIV neutralization in a luciferase    reporter gene assay. Methods Mol Bio 2009, 485:395-405.-   20. Sarzotti-Kelsoe M, Bailer R T, Turk E, Lin C L, Bilska M, Greene    K M, et al. Optimization and validation of the TZM-b1 assay for    standardized assessments of neutralizing antibodies against HIV-1. J    Immunol Methods 2014, 409:131-146.-   21. Edwards et al., J. Virology, 2002, 76:2683-2691.

1. A nucleic acid encoding a synthetic HIV envelope protein comprisingthe amino acid sequence of SEQ ID NO: 8, or SEQ ID NO:8 having one ormore mutations selected from the group consisting of (i) I529P, (ii)K480E, and (iii) a combination of EK479-480RRRR, I529P, A471C and T575C.2. The nucleic acid of claim 1, wherein the synthetic HIV envelopeprotein further comprises a signal sequence.
 3. The nucleic acid ofclaim 1, wherein the synthetic HIV envelope protein further comprises atransmembrane domain.
 4. The nucleic acid of claim 3, wherein thesynthetic HIV envelope protein further comprises a fragment of acytoplasmic domain.
 5. The nucleic acid of claim 1, wherein thesynthetic HIV envelope protein comprises the amino acid sequence of SEQID NO:
 18. 6. The nucleic acid of claim 1, wherein the synthetic HIVenvelope protein either: (a) further comprises a trimerization domain;or (b) comprises SEQ ID NO:8 having a combination of mutationsEK479-480RRRR, I529P, A471C and T575C.
 7. The nucleic acid of claim 6,wherein the synthetic HIV envelope protein comprises the amino acidsequence of residues 1-686 of SEQ ID NO:
 19. 8. A vector comprising thenucleic acid of claim 1, wherein the nucleic acid is operably linked toa promoter sequence.
 9. The vector of claim 8, wherein the vector is anadenovirus vector.
 10. The vector of claim 9, wherein the adenovirusvector is a human adenovirus serotype 26 (Ad26) vector.
 11. An isolatedcell comprising the vector of claim
 8. 12. A composition comprising animmunogenically effective amount of the vector of claim 8, and acarrier.
 13. A vaccine combination, comprising: (i) a first compositioncomprising an immunogenically effective amount of an adenovirus vectorencoding a synthetic HIV envelope protein comprising the amino acidsequence of SEQ ID NO: 8; (ii) a second composition comprising animmunogenically effective amount of a second adenovirus vector encodingan HIV antigenic polypeptide comprising the amino acid sequence of SEQID NO: 5; and optionally (iii) at least one additional compositioncomprising an immunogenically effective amount of at least one selectedfrom the group consisting of (iiia) a vector encoding at least oneantigenic polypeptide having the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-4, 28 and 29, and (iiib) a polypeptidecomprising an immunogenically effective amount of an isolated HIVantigenic polypeptide having residues 30-708 of the amino acid sequenceof SEQ ID NO: 7, or residues 30-724 of SEQ ID NO: 36, wherein the firstcomposition, second composition and additional composition are presentin the same composition or in one or more different compositions.
 14. Amethod of inducing an immune response against a human immunodeficiencyvirus (HIV) in a subject in need thereof, the method comprisingadministering to the subject the composition of claim
 12. 15. Asynthetic HIV envelope protein comprising the amino acid sequence of:(a) SEQ ID NO:8 or SEQ ID NO:8 having one or more mutations selectedfrom the group consisting of (i) I529P, (ii) K480E, and (iii) acombination of EK479-480RRRR, I529P, A471C and T575C; (b) amino acidresidues 30-704 of SEQ ID NO: 18; or (c) amino acid residues 30-686 ofSEQ ID NO:
 19. 16. The nucleic acid of claim 2, wherein the signalsequence comprises the amino acid sequence selected from the groupconsisting of SEQ ID NO: 9 to SEQ ID NO:
 12. 17. The nucleic acid ofclaim 3, wherein the transmembrane domain comprises SEQ ID NO: 13, andoptionally the synthetic HIV envelope protein further comprises SEQ IDNO: 37 fused to the C-terminus of SEQ ID NO: 8 and the N-terminus of thetransmembrane domain.
 18. The nucleic acid of claim 4, wherein thefragment of the cytoplasmic domain comprises the amino acid sequence ofSEQ ID NO: 14 or residues 1-4 thereof.
 19. The nucleic acid of claim 6,wherein the trimerization domain has the amino acid sequence of SEQ IDNO: 15 or SEQ ID NO:
 16. 20. A vector comprising the nucleic acid ofclaim 5, wherein the nucleic acid is operably linked to a promotersequence.
 21. The vector of claim 20, wherein the vector is a viralvector.
 22. The vector of claim 21, wherein the viral vector is anadenovirus vector.
 23. The vector of claim 22, wherein the adenovirusvector is an Ad26 vector.
 24. An isolated cell comprising the vector ofclaim
 20. 25. An isolated cell comprising the vector of claim
 23. 26. Acomposition comprising an immunogenically effective amount of the vectorof claim 20, and a carrier.
 27. A composition comprising animmunogenically effective amount of the vector of claim 22, and acarrier.
 28. A composition comprising an immunogenically effectiveamount of the vector of claim 23, and a carrier.
 29. The vector of claim8, wherein the vector is a viral vector.
 30. The vaccine combination ofclaim 13, wherein the first composition comprises an immunogenicallyeffective amount of an adenovirus vector encoding a synthetic HIVenvelope protein comprising the amino acid sequence of SEQ ID NO: 18.31. The vaccine combination of claim 13, wherein the adenovirus vectorin the first composition is an Ad26 vector.
 32. The vaccine combinationof claim 30, wherein the adenovirus vector in the first composition isan Ad26 vector.
 33. The vaccine combination of claim 13, wherein thesecond adenovirus vector in the second composition is a second Ad26vector.
 34. The vaccine combination of claim 32, wherein the secondadenovirus vector in the second composition is a second Ad26 vector. 35.A method of inducing an immune response against a human immunodeficiencyvirus (HIV) in a subject in need thereof, the method comprisingadministering to the subject the composition of claim
 26. 36. A methodof inducing an immune response against a human immunodeficiency virus(HIV) in a subject in need thereof, the method comprising administeringto the subject the composition of claim
 27. 37. A method of inducing animmune response against a human immunodeficiency virus (HIV) in asubject in need thereof, the method comprising administering to thesubject the composition of claim
 28. 38. A method of inducing an immuneresponse against a human immunodeficiency virus (HIV) in a subject inneed thereof, the method comprising administering to the subject thevaccine combination of claim
 13. 39. A method of inducing an immuneresponse against a human immunodeficiency virus (HIV) in a subject inneed thereof, the method comprising administering to the subject thevaccine combination of claim
 30. 40. A method of inducing an immuneresponse against a human immunodeficiency virus (HIV) in a subject inneed thereof, the method comprising administering to the subject thevaccine combination of claim 32.